[go: up one dir, main page]

EP2325304A1 - ADN polymérases modifiées pour une meilleure intégration d'analogues de nucléotides - Google Patents

ADN polymérases modifiées pour une meilleure intégration d'analogues de nucléotides Download PDF

Info

Publication number
EP2325304A1
EP2325304A1 EP10180445A EP10180445A EP2325304A1 EP 2325304 A1 EP2325304 A1 EP 2325304A1 EP 10180445 A EP10180445 A EP 10180445A EP 10180445 A EP10180445 A EP 10180445A EP 2325304 A1 EP2325304 A1 EP 2325304A1
Authority
EP
European Patent Office
Prior art keywords
polymerase
amino acid
incorporation
polymerases
region
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP10180445A
Other languages
German (de)
English (en)
Other versions
EP2325304B1 (fr
Inventor
Geoffrey Paul Smith
David Mark Dunstan Bailey
David James Earnshaw
Raquel Maria Sanches
Harold Swerdlow
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Illumina Cambridge Ltd
Original Assignee
Illumina Cambridge Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=29226911&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=EP2325304(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Illumina Cambridge Ltd filed Critical Illumina Cambridge Ltd
Publication of EP2325304A1 publication Critical patent/EP2325304A1/fr
Application granted granted Critical
Publication of EP2325304B1 publication Critical patent/EP2325304B1/fr
Anticipated expiration legal-status Critical
Revoked legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

  • the present invention relates to polymerase enzymes and more particularly modified DNA polymerases which give improved incorporation of modified nucleotides compared to a control polymerase. Also included in the present invention are methods of using the polymerases for DNA sequencing, including use of high density arrays.
  • DNA polymerases are relied upon by all organisms to replicate and maintain their genomes. They allow high fidelity replication of DNA by detecting complementarity between bases as well as recognising additional structural features of the base.
  • DNA polymerases Early experiments with DNA polymerases revealed difficulties incorporating modified nucleotide such as dideoxynucleotides (ddNTPs). There are, therefore, several examples in which DNA polymerases have been modified to increase the rates of incorporation of nucleotide analogues. The majority of these have focused on variants of Family A polymerases (eg. Taq) with the aim of increasing the incorporation of dideoxynucleotide chain terminators.
  • Family A polymerases eg. Taq
  • DNA polymerases In order to increase the efficiency of incorporation of modified nucleotides, DNA polymerases have been utilised or engineered such that they lack 3'-5' exonuclease activity (designated exo-).
  • exo- The exo- variant of 9°N polymerase is described by Perler et al., 1998 US 5756334 and by Southworth et al., 1996 Proc. Natl Acad. Sci USA 93:5281 .
  • polymerases have been modified to incorporate nucleotides lacking the hydroxyl group at the 3' carbon of the ribose or deoxyribose sugar moiety.
  • the inventors have now surprisingly found that incorporation of a modified nucleotide having a 3' substituent which is larger that the natural 3' hydroxyl group can be achieved by modified DNA polymerases.
  • the present inventors have devised a method for sequencing DNA that uses nucleotide analogues bearing modifications at the 3' sugar hydroxyl group which block incorporation of further nucleotides.
  • the use of nucleotides bearing a 3' block allows successive nucleotides to be incorporated into a polynucleotide chain in a controlled manner. After each nucleotide addition the presence of the 3' block prevents incorporation of a further nucleotide into the chain. Once the nature of the incorporated nucleotide has been determined, the block may be removed, leaving a free 3' hydroxyl group for addition of the next nucleotide.
  • DNA polymerases which can incorporate efficiently nucleotides which are modified at the 3' sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3' hydroxyl group.
  • modification of the 3' sugar hydroxyl group results in substrates that terminate DNA synthesis.
  • nucleotides that bear modifications at the 3' sugar position that, under appropriate conditions, can be reacted to form a 3' hydroxyl group. These nucleotides are referred to as ⁇ reversible terminators'.
  • DNA polymerases that have the ability to incorporate such 3' blocked nucleotides have applications that involve DNA synthesis, such as DNA sequencing.
  • the altered DNA polymerases must be able to retain base specificity for the modified nucleotides, even though efficiency of incorporation is increased.
  • certain cofactors such as Manganese ions, have been shown to increase nucleotide misincorporation by DNA polymerases; however, for certain applications, such as DNA sequencing, it is important that reaction conditions are chosen that allow the fidelitous incorporation of modified nucleotides. In these cases, it would be preferable to not include Manganese or other cofactors that increase misincorporation into the reaction mixtures.
  • the inventors have shown that the ability to incorporate the modified nucleotides can be conferred by as little as one amino acid change in the nucleotide binding site of the enzyme. It is extremely surprising that such a profound effect on the substrate specificity of the enzyme can be achieved at all, but it is even more unexpected that such an effect can be achieved with such a limited change in primary amino acid sequence.
  • altered polymerases are capable of incorporating modified nucleotides containing all four natural DNA bases A, T, C and G.
  • This again is an unexpected finding, as one skilled in the art might reasonably expect that different modified polymerases would have to be engineered to incorporate analogues containing different bases. This in turn would mean that a cocktail of different polymerases, possibly requiring different reaction conditions for optimal activity, would be needed in order to perform a sequencing reaction.
  • the surprising finding that altered polymerases can be constructed such that a single enzyme is capable of incorporating modified nucleotide analogues containing each of the four natural DNA bases therefore provides a huge advantage in sequencing protocols, not least because all four bases can be incorporated under the same reaction conditions.
  • Altered polymerases of the invention also exhibit activity over a wide temperature range, and more specifically exhibit activity at low temperatures far removed from the temperature optimum of the equivalent wild type enzymes.
  • the inventors have shown that altered polymerases are active at temperatures as low as 45°, and even as low as 30°C, which is far removed from the temperature optimum of 80°C for the 9°N polymerase.
  • altered polymerases are suitable for use in sequencing protocols carried out at low and high temperatures. This finding is again extremely unexpected, since one might reasonably expect that in order to observe an acceptable rate of incorporation of modified nucleotide analogues it would be necessary to work at conditions close to the optimum for activity of the wild-type enzyme, e.g. a temperature of 80°C for the 9°N polymerase.
  • modified nucleotide analogue substrate required to achieve incorporation using altered polymerases of the invention is much lower than had been anticipated.
  • concentration of modified nucleotide analogue substrate is much lower than had been anticipated.
  • One skilled in the art might have expected that a high concentration of modified nucleotide would be required in order to achieve incorporation, effectively to "force" the enzyme to incorporate the modified nucleotide.
  • the ability to use lower concentrations of substrate is important as it leads to reduced non-specific binding as well as being more economical.
  • Altered polymerase enzymes exhibit improved incorporation of the modified nucleotides over multiple enzyme cycles. This property is again important for sequencing-by-synthesis applications requiring sequential incorporation of nucleotides into a polynucleotide chain.
  • An enzyme that can incorporate modified nucleotides in one cycle of enzymology only would be of limited value in sequencing because different polymerases would need to be added at different cycles.
  • modified polymerases that have been identified for the incorporation of nucleotides that bear large 3' substituents would also be capable of incorporating natural nucleotides and other nucleotide analogues.
  • the inventors have produced altered polymerases which are capable of incorporating modified nucleotides bearing large 3' substituents and which exhibit a number of advantageous properties.
  • an altered polymerase enzyme which is capable of improved incorporation of nucleotides which have been modified at the 3' sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3' hydroxyl group, compared to a control polymerase) enzyme.
  • incorporation means joining of the modified nucleotide to the free 3' hydroxyl group of a second nucleotide via formation of a phosphodiester linkage with the 5' phosphate group of the modified nucleotide.
  • the second nucleotide to which the modified nucleotide is joined will typically occur at the 3' end of a polynucleotide chain.
  • modified nucleotides and “nucleotide analogues” when used in the context of this invention refer to nucleotides which have been modified at the 3' sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3' hydroxyl.group. These terms may be used interchangeably.
  • large 3' substituent(s) refers to a substituent group at the 3' sugar hydroxyl which is larger in size than the naturally occurring 3' hydroxyl group.
  • Improved incorporation is defined herein to include an increase in the efficiency and/or observed rate of incorporation of at least one modified nucleotide, compared to a control polymerase enzyme.
  • the invention is not limited just to improvements in absolute rate of incorporation of the modified nucleotides.
  • the altered polymerases exhibit other desirable properties related to the incorporation of nucleotide analogues and the term "improved incorporation” is to be interpreted accordingly as also encompassing improvements in any of these other properties, with or without an increase in the rate of incorporation.
  • the improvement in the efficiency and/or observed rate of incorporation may be manifest on any cycle of enzymology, but may occur on two or more cycles, and most preferably on all cycles.
  • the "improvement" need not be constant over all cycles.
  • the altered polymerase may exhibit improvement in the efficiency and/or observed rate of incorporation for one type of modified nucleotide or for a range of different modified nucleotides.
  • the polymerase may be capable of incorporating a series of modified nucleotides which have the same 3' substituent but which differ in some other portion of the nucleotides molecule, such as the base portion or the presence of a detectable label.
  • Other polymerases may be capable of incorporating a range of modified nucleotides which carry different 3' substituents.
  • Improvements in the efficiency and/or rate of incorporation achieved with the altered polymerase relative to a control polymerase may be determined by comparing the ability of the two polymerases to incorporate the same modified nucleotide(s) in a suitable assay system.
  • the ability of the altered polymerase to incorporate modified nucleotide(s) may also be defined by reference to an absolute measure of incorporation in a specified assay system.
  • One such assay system given by way of example, is the gel-based incorporation assay described in the accompanying Example 2.
  • Preferred polymerases exhibiting the ability to incorporate modified nucleotides are those which show detectable (i.e.
  • the polymerase may exhibit a time to 50% product conversion on the second cycle of enzymology at 45°C, 50mM Tris, pH8, 4mM MgSO 4 , 0.05 (v/v) Tween, 50 ⁇ g/ml enzyme, and 2 ⁇ M 3' modified nucleotide of 19.5 min or less, more preferably 5 min or less and most preferably 1.5 min or less.
  • product conversion on the first cycle will be less than 1 minute under these conditions, as described in Example 2.
  • the enhanced ability of the altered polymerase to incorporate modified nucleotides may also be determined by measurement of incorporation rates at different nucleotide concentrations. Altered polymerases exhibiting the desired "improved activity" will preferably exhibit a greater than 10% nucleotide incorporation at a concentration of 25 ⁇ M.
  • the invention is not limited to solely to altered polymerases that show improvements in enzyme kinetics.
  • the invention also encompasses altered polymerase enzymes which exhibit other "improvements" in incorporation of modified nucleotides which are of practical and economic benefit.
  • the "improvement" exhibited by the altered polymerase may be the ability to incorporate modified nucleotides containing each of the natural DNA bases A, T, C and G.
  • the ability to incorporate all four bases with a single enzyme is of great practical and economic value in sequencing protocols. Even if absolute rates of incorporation were comparable between, for example, four different enzymes each having specificity for modified nucleotides containing a different base A, T, C or G, and a single enzyme capable of incorporating nucleotides containing each of the four bases, there would be considerable practical advantages in the use of a single enzyme capable of incorporating all four bases. Use of a single enzyme avoids any problems in providing optimum reaction conditions for different polymerases.
  • the "improvement" may be the ability to incorporate the modified nucleotides at low temperatures and/or over a wider temperature range than the control enzyme.
  • the altered polymerases of the invention are capable of incorporating modified nucleotides at a temperature of 45°C, and even as low as 30°C.
  • Preferred polymerases are those which exhibit detectable incorporation of the modified nucleotides at a temperature of 30°C and/or at a temperature of 45°C.
  • the enzyme will exhibit detectable incorporation of the modified nucleotide over a range of temperatures above and/or below 30°C or 45°C.
  • the polymerase may exhibit detectable incorporation activity across the whole range of temperatures from 30°C or 45°C up to the optimum temperature for activity of the equivalent wild-type (or exo-) enzyme, which could be as high as 80°C for enzymes derived from thermophilic species.
  • Preferred polymerases may exhibit detectable incorporation activity across the whole range of temperatures from 30°C to 37°C or from 30°C to 45°C and/or from 65°C to 80°C.
  • the rate of incorporation may not be constant over the full range of temperature over which the enzyme exhibits detectable incorporation activity and incorporation rate need not be maximal at 30°C or 45°C. Even if the absolute rate of incorporation of a given modified nucleotide were comparable between, for example, the altered polymerase working at low temperature (e.g. 30°C or 45°C) and wild type polymerase working close to the natural optimum temperature for enzyme activity (e.g. 80°C for 9°N polymerase) it would still be of great practical benefit to be able to work at the lower temperature because it reduces the requirement for other components in the sequencing system to be tolerant to the high temperature conditions.
  • the altered polymerase working at low temperature e.g. 30°C or 45°C
  • wild type polymerase working close to the natural optimum temperature for enzyme activity
  • the "improvement" may be the ability to incorporate the modified nucleotides when using a lower concentration of the modified nucleotides as substrate.
  • the altered polymerase should exhibit detectable incorporation of the modified nucleotide when working at a substrate concentration in the micromolar range.
  • Preferred polymerases may exhibit detectable incorporation of the modified nucleotide when working at a substrate concentration in the range of from 0.2 ⁇ M to 50 ⁇ M, and more preferably at a substrate concentration of 25 ⁇ M.
  • the polymerases of the-invention may exhibit detectable incorporation of the modified nucleotide when working at substrate concentrations below 0.2 ⁇ M (and/or in excess of 50 ⁇ M).
  • Certain polymerases according to the invention exhibit detectable incorporation of the modified nucleotide when working at substrate concentrations as low as 50nM.
  • altered polymerase enzyme it is meant that the polymerase has at least one amino acid change compared to the control polymerase enzyme. In general this change will comprise the substitution of at least one amino acid for another. In certain instances these changes will be conservative changes, to maintain the overall charge distribution of the protein. However, the invention is not limited to only conservative substitutions. Non-conservative substitutions are also envisaged in the present invention.
  • the modification in the polymerase sequence may be a deletion or addition of one or more amino acids from or to the protein, provided that the polymerase has improved activity with respect to the incorporation of nucleotides modified at the 3' sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3' hydroxyl group as compared to a control polymerase enzyme.
  • Nucleotide is defined herein to include both nucleotides and nucleosides. Nucleosides, as for nucleotides, comprise a purine or pyrimidine base linked glycosidically to ribose or deoxyribose, but they lack the phosphate residues which would make them a nucleotide. Synthetic and naturally occurring nucleotides, prior to their modification at the 3' sugar hydroxyl, are included within the definition.
  • the altered polymerase will generally be an "isolated” or “purified” polypeptide.
  • isolated polypeptide is meant a polypeptide that is essentially free from contaminating cellular components, such as carbohydrates, lipids, nucleic acids or other proteinaceous impurities which may be associated with the polypeptide in nature.
  • a preparation of the isolated polymerase contains the polymerase in a highly purified form, i.e. at least about 80% pure, preferably at least about 90% pure, more preferably at least about 95% pure, more preferably at least about 98% pure and most preferably at least about 99% pure. Purity of a preparation of the enzyme may be assessed, for example, by appearance of a single band on a standard SDS-polyacrylamide gel electrophoresis.
  • the altered polymerase may be a "recombinant" polypeptide.
  • the altered polymerase according to the invention may be a family B type DNA polymerase, or a mutant or variant thereof.
  • Family B DNA polymerases include numerous archael DNA polymerase, human DNA polymerase ⁇ and T4, RB69 and ⁇ 29 phage DNA polymerases. These polymerases are less well studied than the family A polymerases, which include polymerases such as Taq, and T7 DNA polymerase.
  • the polymerase is selected from any family B archael DNA polymerase, human DNA polymerase ⁇ or T4, RB69 and ⁇ 29 phage DNA polymerases.
  • the archael DNA polymerases are in many cases from hyperthermophilic archea, which means that the polymerases are often thermostable. Accordingly, in a further preferred embodiment the polymerase is selected from Vent, Deep Vent, 9°N and Pfu polymerase. Vent and Deep Vent are commercial names used for family B DNA polymerases isolated from the hyperthermophilic archaeon Thermococcus litoralis. 9°N polymerase was also identified from Thermococcus sp. Pfu polymerase was isolated from Pyrococcus furiosus. The most preferred polymerase in the present invention is 9°N polymerase, including mutants and variants thereof.
  • the altered polymerase may also be a family A polymerase, or a mutant or variant thereof, for example a mutant or variant Taq or T7 DNA polymerase enzyme, or a polymerase not belonging to either family A or family B, such as for example reverse transcriptases.
  • Control polymerase is defined herein as the polymerase against which the activity of the altered polymerase is compared.
  • the control polymerase may comprise a wild type polymerase or an exo- variant thereof. Unless otherwise stated, by “wild type” it is generally meant that the polymerase comprises its natural amino acid sequence, as it would be found in nature.
  • control polymerase can, therefore, comprise any known polymerase, including mutant polymerases known in the art.
  • the (in)activity the chosen "control" polymerase with respect to incorporation of the desired nucleotide analogues may be determined by an incorporation assay.
  • Preferred control polymerases are those which exhibit no detectable incorporation of modified nucleotides in the incorporation assay described in Example 2.
  • control polymerases having one or more amino acid substitution mutations relative to the sequence of a wild-type (or exo- variant) base polymerase are summarised below: Table I - examples of control polymerases Base polymerase (WT or exo-) Additional mutations functionally equivalent to amino acid sequence of the base polymerase VentTM Y412V, Y412L, Y412F, Q486E, Q486L, R487K, A488C, A488S, A488L, A488I, A488F, A488V, K490A, FC490R, K490N, N494D, S495A, Y496F, Y496L, Y497S, Y497F, Y499L, Y499F, A488C/Y499F or A488L/Y499L. 9°N A485L or Y409V/A485L (latter is utilised
  • the control polymerase may comprises any one of the listed substitution mutations functionally equivalent to the amino acid sequence of the given base polymerase (or an exovariant thereof).
  • the control polymerase may be a mutant version of the listed base polymerase having one of the stated mutations or combinations of mutations, and preferably having amino acid sequence identical to that of the base polymerase (or an exo- variant thereof) other than at the mutations recited above.
  • the control polymerase may be a homologous mutant version of a polymerase other than the stated base polymerase, which includes a functionally equivalent or homologous mutation (or combination of mutations) to those recited in relation to the amino acid sequence of the base polymerase.
  • control polymerase could be a mutant version of the VentTM polymerase having one of the mutations or combinations of mutations listed above relative to the VentTM amino acid sequence, or it could be a mutant version of another polymerase, for example a mutant 9°N polymerase, incorporating a mutation (or mutations) functionally equivalent to a mutation (or mutations) listed by reference to the VentTM amino acid sequence.
  • control polymerase in the case of studies using a different polymerase entirely, will contain the amino acid substitution that is considered to occur at the amino acid position in the other polymerase that has the same functional role in the enzyme.
  • the mutation at position 412 from Tyrosine to Valine (Y412V) in the Vent DNA polymerase would be functionally equivalent to a substitution at position 409 from Tyrosine to Valine (Y409V) in the 9°N polymerase.
  • the bulk of this amino acid residue is thought to act as a "steric gate" to block access of the 2'-hydroxyl of the nucleotide sugar to the binding site.
  • residue 488 in Vent polymerase is deemed equivalent to amino acid 485 in 9°N polymerase, such that the Alanine to Leucine mutation at 488 in Vent (A488L) is deemed equivalent to the A485L mutation in 9°N polymerase.
  • This residue is thought to play a role in changing the activation energy required for the enzymatic reaction.
  • Functionally equivalent, positionally equivalent and homologous amino acids within the wild type amino acid sequences of two different polymerases do not necessarily have to be the same type of amino acid residue, although functionally equivalent, positionally equivalent and homologous amino acids are commonly conserved.
  • the motif A region of 9°N polymerase has the sequence LYP
  • the functionally homologous region of VentTM polymerase also has sequence LYP.
  • the homologous amino acid sequences are identical, however homologous regions in other polymerases may have different amino acid sequence.
  • mutant amino acid When referring to functionally equivalent, positionally equivalent and homologous mutations, the mutant amino acid will generally be the same type of amino acid residue in each of the mutant polymerases, for example mutation Y409A in 9°N polymerase is functionally equivalent or homologous to mutation Y412A in VentTM polymerase.
  • the inventors have surprisingly determined that amino acid sequence variation in a particular three amino-acid region of the nucleotide binding site referred to herein as the "motif A region" has a profound effect on the ability of polymerases to incorporate nucleotide analogues having a substituent at the 3' position which is larger than a hydroxyl group.
  • motif A region specifically refers to the three amino acids functionally equivalent or homologous to amino acids 408-410 in 9°N polymerase.
  • motif A region and region A are used interchangeably throughout and refer to the defined three amino acid region of the polymerase protein rather than to any particular amino acid sequence.
  • motif B region specifically refers to the three amino acids functionally equivalent or homologous to amino acids 484-486 in 9°N polymerase.
  • motif A and motif B have been used in the art in order to refer to regions of sequence homology in the nucleotide binding sites of family B and other polymerases. However, in the context of this application these terms are used to refer only to the specific amino acid regions recited above.
  • Mutation of the motif A region alone, in the absence of any other sequence variation elsewhere in the enzyme, is sufficient to substantially improve the ability of the polymerase to incorporate nucleotide analogues having a substituent at the 3' position which is larger than a hydroxyl group.
  • a substitution mutation of a single amino acid in the motif A region of a polymerase can be sufficient to improve the ability of the polymerase to incorporate nucleotides which have been modified at the 3' sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3' hydroxyl group.
  • Substitution mutations in two or all three of the amino acids in the motif A region can also improve the ability of a polymerase to incorporate the desired analogues.
  • the preferred altered polymerases according to the invention are mutant polymerases comprising one, two or three amino acid substitution mutations in the three-amino acid region of the polymerase referred to herein as the "motif A region".
  • the substitution mutation(s) may lead to the incorporation of any known natural or synthetic amino acid, subject to the following provisos:
  • the specific polymerases recited under provisos (1), (2) and (3) are excluded from the invention. It is preferred that the altered polymerase is not a different type of polymerase bearing functionally equivalent or homologous amino acid changes to the polymerases recited under provisos (1), (2) and (3), although such homologous polymerases are not excluded.
  • the inventors have determined that variation of the amino acid sequence in the motif A region alone can result in improvements in the desired activity of the polymerase, substantially improving the rate of incorporation of nucleotide analogues having large substituents at the 3' position. Moreover, the inventors have determined consensus sequence preferences for the three amino acids making up the motif A region.
  • first amino acid of the motif A region is meant the amino acid functionally equivalent or homologous to amino acid residue 408 of the 9°N polymerase.
  • second amino acid of the motif A region is meant the amino acid functionally equivalent or homologous to amino acid residue 409 of the 9°N polymerase.
  • the second amino acid of the motif A region is preferably selected from alanine (A), serine (S) or glycine(G), most preferably alanine (A).
  • this amino acid is preferably not valine (V), leucine (L) or phenylalanine (F).
  • valine (V), leucine (L) or phenylalanine (F) substitution to valine (V), leucine (L) or phenylalanine (F) may be present in combination with further substitutions at the first and/or third amino acid positions in the motif A region.
  • third amino acid of the motif A region is meant the amino acid functionally equivalent or homologous to amino acid residue 410 of the 9°N polymerase.
  • the third amino acid of the motif A region is preferably selected from:
  • the substitution may be present at the first, second or third amino acid position of the motif A region, subject to the provisos given above.
  • the single amino acid substitution it is preferred for the single amino acid substitution to be present at the second amino acid position, this being the amino acid functionally equivalent or homologous to amino acid residue 409 of the 9°N polymerase.
  • the mutant amino acid will preferably be selected according to the amino acid preferences recited above for the first, second and third amino acids of the motif A region.
  • Particularly preferred polymerases having a single substitution mutation in the motif A region are those wherein the second amino acid residue of the motif A region is mutated to alanine (A). Therefore, preferred mutant polymerase include, but are not limited to, 9°N polymerase having the mutation Y409A, VentTM polymerase having the mutation Y412A, Pfu polymerase having the mutation Y410A, JDF-3 polymerase having the mutation Y409A, and Taq polymerase having the mutation E615A.
  • polymerases having a single amino acid substitution mutation in the motif A region may include further substitution mutations compared to wild type in regions of the enzyme outside of the motif A region within the scope of the invention.
  • the mutations may be present in any combination, i.e. the first and second amino acids may be mutant, or the first and third amino acids may be mutant, or the second and third amino acids may be mutant. It is generally preferred for the first and second amino acids of the motif A region to be mutant, with the third amino acid being wild-type.
  • the mutant amino acids will again preferably be selected according to the amino acid preferences recited above for the first, second and third amino acids of the motif A region. All possible combinations of two of the amino acid preferences given above for the first, second and third amino acids are envisaged within the scope of the invention.
  • particularly preferred polymerases having two substitution mutations are those wherein the first amino acid residue of the motif A region is mutated to valine (V), phenylalanine (F) or tyrosine (Y), the second amino acid residue of the motif A region is mutated to alanine (A) and the third amino acid residue of the motif A region is wild type.
  • preferred mutant polymerase include, but are not limited to, 9°N polymerase having one of double mutations L408F/Y409A, L408V/Y409A or L408Y/Y409A, VentTM polymerase having one of double mutations L411Y/Y412A, L411V/Y412A or L411F/Y412A, Pfu polymerase having one of double mutations L409Y/Y410A, L409V/Y410A or L409F/Y410A, JDF-3 polymerase having one of double mutations L408F/Y409A, L408Y/Y409A or L408V/Y409A, and Taq polymerase having one of double mutations I614F/E615A, I709V/E710A or I614Y/E615A.
  • polymerases having two amino acid substitution mutations in the motif A region may include further substitution mutations compared to wild type in regions of the enzyme outside of the motif A region within the scope of the invention.
  • Altered polymerases according to the invention may also include three amino acid substitution mutations in the motif A region.
  • the mutant amino acids will again preferably be selected according to the amino acid preferences recited above for the first, second and third amino acids of the motif A region. All possible combinations of three of the amino acid preferences given above for the first, second and third amino acids are envisaged within the scope of the invention.
  • the motif A region of the polymerase may have one of the following mutant amino acid sequences:
  • modified DNA polymerases incorporating these region A sequences show substantially improved incorporation of nucleotides which have been modified at the 3' sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3' hydroxyl group, compared to the 9°N double mutant.
  • the most effective mutants are those having the region A sequences YAV and YAS. Therefore, preferred polymerases according to the invention are those having amino acid sequence YAV or YAS in the motif A region.
  • Most preferred mutant polymerase include, but are not limited to, 9°N polymerase having one of the triple mutations L408Y/Y409A/P410S or L408Y/Y409A/P410V, VentTM polymerase having one of the triple mutations L411Y/Y412A/P413V or L411Y/Y412A/P413S, Pfu polymerase having one of the triple mutations L409Y/Y410A/P411V or L409Y/Y410A/P411S, JDF-3 polymerase having one of the triple mutations L408Y/Y409A/P410V or L408Y/Y409A/P410S, and Taq polymerase having one of the triple mutations I614Y/E615A/L616V or I614Y/E615A/L616S.
  • polymerases having three amino acid substitution mutations in the motif A region may include further substitution mutations compared to wild type in regions of the enzyme outside of the motif A region within the scope of the invention.
  • the polymerases exhibiting one, two or three amino acid substitution mutations in the motif A region may additionally comprise one, two or three substitution mutations in the motif B region. It has been shown that certain region B mutations, in combination with region A mutations, can still further enhance the activity of the polymerase.
  • the polymerase may include a single amino acid substitution at the first, second or third amino acid residue of the motif B region, or may include any combination of two or more mutations or may be mutated at all three residues in region B.
  • the first amino acid of the motif B region is defined as the amino acid functionally equivalent or homologous to residue 484 of the 9°N polymerase amino acid sequence
  • the second amino acid of the motif B region is defined as the amino acid functionally equivalent or homologous to residue 485 of the 9°N polymerase amino acid sequence
  • the third amino acid of the motif B region is defined as the amino acid functionally equivalent or homologous to residue 486 of the 9°N polymerase amino acid sequence
  • Preferred altered polymerases according to the invention may include substitution mutations at the second amino acid of the motif B region, this being the amino acid functionally equivalent or homologous to residue 485 of the 9°N polymerase amino acid sequence, in addition to sequence variation in the motif A region. Mutation of this residue (or its functional equivalent or homologue in polymerases other than 9°N) has generally been observed to enhance overall polymerase activity. Hence, inclusion of a mutation at this position, in combination with region A variation, may produce still further enhancement of activity compared to region A variation alone.
  • substitutions are preferred: A485L, A485F, A485I, A485S, A485V and A485C, with A485L being the most preferred.
  • substitution mutation at the second amino acid of the motif B region may be present singly (meaning in the absence of any other mutation in the motif B region) or may be present with one or two further substitutions in the motif B region.
  • the mutations may occur in any combination, i.e. first and second, first and third or second and third residues mutated. However, it is preferred for the first and third residues to be mutated in combination. Particularly preferred combinations are mutation to glycine at the first amino acid position of the motif B region, in combination with mutation to leucine at the third amino acid position, or mutation to asparagine at the first amino acid position of the motif B region in combination with mutation to glutamine at the third amino acid position.
  • suitable mutant amino acid sequences for the motif B region include, but are not limited to, the following: SKN, GRD, KHN, ISN and THH.
  • the altered polymerase of the invention may include any of the region A mutations or combinations of mutations recited herein either in the absence of any region B mutation or in combination with any of the specific region B mutations or combinations of mutations described herein.
  • the invention encompasses altered polymerases comprising at least one mutation in each of the motif A and motif B regions, wherein the total number of mutations is greater than two.
  • the invention encompasses, although is not limited to, polymerases having two or three mutations in the motif A region with one mutation in the motif B region.
  • Particularly preferred polymerases according to the invention are 9°N polymerases having one of the following motif A region sequences: FAP, VAP, YST, FAI, AAA, YAS, YAV, YGI, YSG, SGG, CST, IAL, CGG, SAL, SAA, CAA, YAA, QAS, VSS, VAG, VAV, FAV, AGI, YSS, AAT, FSS or VAL, most preferably YAV or YAS, plus a substitution mutation at position 485 in region B, most preferably A485L.
  • motif A region sequences FAP, VAP, YST, FAI, AAA, YAS, YAV, YGI, YSG, SGG, CST, IAL, CGG, SAL, SAA, CAA, YAA, QAS, VSS, VAG, VAV, FAV, AGI, YSS, AAT, FSS or VAL, most preferably YAV or YAS,
  • the invention also encompasses polymerases other than 9°N, such as for example VentTM polymerases, Pfu polymerases, JDF-3 polymerases, Taq polymerases etc including homologous amino acid substitutions (list is illustrative, not limiting).
  • the preferred 9°N polymerases recited in the preceding paragraph all exhibit surprisingly improved incorporation of nucleotide analogues which have been modified at the 3' sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3' hydroxyl group compared to the 9°N polymerase double mutant (9°N DM, having mutations Y409V/A485L) control polymerase, as illustrated in the accompanying examples.
  • Polymerases including the motif A region sequences YAV or YAS exhibited the best incorporation rate for the desired analogues of those polymerases tested under the conditions of the Example.
  • the invention also relates to a 9°N polymerase molecule comprising the amino acid sequence shown as SEQ ID NO: 20 and a 9°N polymerase molecule comprising the amino sequence shown as SEQ ID NO: 22.
  • SEQ ID NO: 20 represents the complete amino acid sequence of the 9°N polymerase YAS mutant tested in the experimental section (also includes mutation A485L)
  • SEQ ID NO: 22 represents the complete amino acid sequence of the 9°N polymerase YAV mutant (also including mutation A485L).
  • the invention also encompasses polymerases having amino acid sequences which differ from those shown as SEQ ID NOs: 20 and 22 only in amino acid changes which do not affect the function of the polymerase to a material extent.
  • the relevant function of the polymerase is defined as an improved ability, as compared to the control protein, to incorporate modified nucleotides having a substituent at the 3' sugar hydroxyl which is larger in size than the naturally occurring 3' hydroxyl group.
  • conservative substitutions at residues which are not important for this activity of the YAV or YAS polymerase variants would be included within the scope of the invention.
  • the effect of further mutations on the function of the enzyme may be readily tested, for example using the incorporation assay described in the accompanying examples.
  • the invention also relates to a 9°N polymerase molecule comprising the amino acid sequence shown as SEQ ID NO: 18.
  • SEQ ID NO: 18 represents the complete amino acid sequence of the 9°N polymerase "ED" mutant tested in the experimental section, which includes the motif A region sequence VAL in addition to the A485L mutation in the motif B region.
  • This polymerase variant exhibits improved incorporation of the 3' modified nucleotide analogues having substituents larger than the hydroxyl group as compared to the 9°N polymerase double mutant (Y409V/A485L).
  • the polymerase variant may comprise three amino acid substitution mutations in the motif A region and two or three amino acid substitution mutations in the motif B region.
  • suitable polymerase variants have been discovered by introducing mutations simultaneously at the motif A region and the motif B region in the 9°N polymerase.
  • a library of polymerase variants was screened for the incorporation of the 3' modified nucleotide analogues. Variants that displayed enhanced levels of incorporation compared to the parental clone were purified and tested to confirm their activity.
  • the invention also provides, but is not limited to, an altered polymerase which comprises any one of the following sets of substitution mutations functionally equivalent or homologous to the 9°N DNA polymerase amino acid sequence:
  • control polymerase can be the wild type 9°N polymerase or alternatively a substituted polymerase, such as the Y409V/A485L double mutant 9°N polymerase (9DM).
  • the invention also relates to a 9°N polymerase molecule comprising the amino sequence as shown in SEQ ID NO: 12.
  • This SEQ ID NO: 12 represents the amino acid sequence of the 2E protein.
  • the invention also encompasses polymerases having amino acid sequences which differ from that shown as SEQ ID NO: 12 only in amino acid changes which do not affect the function of the polymerase to a material extent. In this case the relevant function would be the improved ability, as compared to the control protein, of the polymerase to incorporate modified nucleotides. Thus conservative substitutions at residues which are not important for this activity of the 2E polymerase would be included within the scope of the invention.
  • the invention provides a 9°N polymerase comprising the amino acid sequence as set out in SEQ ID NO: 14 (sequence of 9°N polymerase lacking exonuclease activity) with one or more changes in the amino acid sequence between residues 408 and 410 and optionally between residues 484 and 486, excluding changes which would result in a protein with a valine residue at amino acid 409 and a leucine residue at amino acid 485, or a protein with a leucine residue at amino acid 485, in the absence of any other changes in the amino acid sequence between residues 408 and 410 or between residues 484 and 486.
  • amino acid changes introduced into SEQ ID No: 14 must be such that they do not only alter residue 409 to valine and residue 485 to leucine or alter amino acid 485 to leucine. These changes can occur, but the must not occur together (double mutant) or singly (leucine at position 485) in isolation from a further change in the amino acid sequence of the 9°N polymerase. A single amino acid change can be sufficient to improve functionality of 9°N in terms of incorporation of modified nucleotides.
  • the change in the amino acid sequence may incorporate any known natural or synthetic amino acid.
  • the altered 9°N polymerases will catalyse improved incorporation of nucleotides which have been modified at the 3' sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3' hydroxyl group.
  • Preferred embodiments of all of the above-described polymerase variants having mutations in the motif A region alone or in combination with mutations in the motif B region have wild-type sequence in the motif C region of the nucleotide binding site.
  • the "motif C region” is defined herein as the amino acid residues functionally equivalent or homologous to residues 493, 494 and 496 of the 9°N polymerase amino acid sequence.
  • variants having one or more amino acid changes in this region in addition to changes in the motif A region, with or without changes in the motif B region are not excluded.
  • variants having conservative amino acid changes in the motif C region are included within the scope of the invention.
  • the altered polymerases of the invention may include further changes in amino acid sequence, as compared to the equivalent wild-type protein. Further amino acid substitutions, deletions, additions, fusions etc. may be incorporated, for example in order to introduce a desirable property into the enzyme and/or to improve recombinant expression and/or to facilitate purification of the protein and/or in order to remove an undesirable property of the enzyme.
  • the altered polymerases may also incorporate amino acid changes which, in themselves, do not have a significant or detectable effect on polymerase activity and which do not alter any other function of the polymerase or confer any desirable property. These may include, for example, conservative amino acid substitutions in regions of the protein outside of the motif A region and motif B region.
  • the altered polymerase enzyme of the invention may be further altered such that it lacks 3'-5' exonuclease activity.
  • This 3'-5' exonuclease activity is absent in certain DNA polymerases such as Taq DNA polymerase. It is useful to remove this exonuclease proof-reading activity when using modified nucleotides to prevent the exonuclease removing the non-natural nucleotide after incorporation.
  • Such changes to DNA polymerase enzymes is therefore of benefit for DNA sequencing applications involving 3' modified nucleotides.
  • Archael DNA polymerases can be genetically modified within the conserved exonuclease motif, changing the amino acid sequence from DIE to AIA, in order to remove 3'-5' exonuclease activity.
  • Such changes have previously been made for the Vent and Deep Vent DNA polymerases (New England Biolabs; see Nucleic Acids, Research 1999, Vol. 27, No. 12 and Nucleic Acids Research, 2002, Vol. 30, No. 2 ).
  • In the 9°N polymerase exonuclease activity may be removed by including the substitution mutations D141A and E143A.
  • the invention further relates to nucleic acid molecules encoding the altered polymerase enzymes of the invention.
  • nucleotide sequence encoding the mutant For any given altered polymerase which is a mutant version of a polymerase for which the amino acid sequence and preferably also the wild type nucleotide sequence encoding the polymerase is known, it is possible to obtain a nucleotide sequence encoding the mutant according to the basic principles of molecular biology. For example, given that the wild type nucleotide sequence encoding 9°N polymerase is known, it is possible to deduce a nucleotide sequence encoding any given mutant version of 9°N having one or more amino acid substitutions using the standard genetic code. Similarly, nucleotide sequences can readily be derived for mutant versions other polymerases such as, for example, VentTM, Pfu, Tsp JDF-3, Taq, etc. Nucleic acid molecules having the required nucleotide sequence may then be constructed using standard molecular biology techniques known in the art.
  • the invention relates to nucleic acid molecules encoding mutant versions of the 9°N polymerase. Therefore, the invention provides a nucleic acid molecule comprising the nucleotide sequence shown as SEQ ID NO: 13 but having one or more changes in the nucleic acid sequence in the region from nucleotide 1222 to nucleotide 1230 (inclusive of these nucleotides), wherein said nucleic acid molecule encodes a mutant 9°N polymerase enzyme having one, two or three amino acid substitution mutations in the region from amino acid residue 408 to amino acid residue 410, with the proviso that the nucleic acid molecule does not encode a mutant 9°N polymerase having the amino acid substitution mutations Y409V and A485L in combination.
  • the nucleotide sequence may optionally further include one or more changes in the region from nucleotide 1449 to nucleotide 1457 (inclusive of these residues) as compared to the sequence shown as SEQ ID NO:13, such that the mutant 9°N polymerase enzyme polymerase enzyme further includes one, two or three amino acid substitution mutations in the region from amino acid residue 484 to residue 486.
  • Changes in the nucleic acid sequence shown as SEQ ID NO: 13 according to this aspect of the invention must be such that they alter the amino acid sequence of the polymerase. Changes in the nucleic acid sequence that are silent with respect to the overall amino acid sequence of the 9°N polymerase are not included in this aspect of the invention.
  • the change of amino acid residue 409 to valine and residue 485 to leucine, or the change of amino acid 485 to a leucine residue should not be the only changes that are made to the polymerase. These changes can occur, but they must not occur together (double mutant) in the absence of any further change in the amino acid sequence of the 9°N polymerase other then the exo- mutations D141A and E143A.
  • the invention provides a nucleic acid molecule encoding a mutant 9°N polymerase, the nucleic acid molecule comprising one of the following the nucleotide sequences:
  • nucleic acid sequence also includes the complementary sequence to any single stranded sequence given regarding base variations.
  • nucleic acid molecules described herein may also, advantageously, be included in a suitable expression vector to express the polymerase proteins encoded therefrom in a suitable host. Incorporation of cloned DNA into a suitable expression vector for subsequent transformation of said cell and subsequent selection of the transformed cells is well known to those skilled in the art as provided in Sambrook et al. (1989), Molecular cloning: A Laboratory Manual, Cold Spring Harbour Laboratory .
  • Such an expression vector includes a vector having a nucleic acid according to the invention operably linked to regulatory sequences, such as promoter regions, that are capable of effecting expression of said DNA fragments.
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • Such vectors may be transformed into a suitable host cell to provide for the expression of a protein according to the invention.
  • the nucleic acid molecule may encode a mature protein or a protein having a prosequence, including that encoding a leader sequence on the preprotein which is then cleaved by the host cell to form a mature protein.
  • the vectors may be, for example, plasmid, virus or phage vectors provided with an origin of replication, and optionally a promoter for the expression of said nucleotide and optionally a regulator of the promoter.
  • the vectors may contain one or more selectable markers, such as, for example, an antibiotic resistance gene.
  • a bacterial expression vector may include a promoter such as the lac promoter and for translation initiation the Shine-Dalgarno sequence and the start codon AUG.
  • a eukaryotic expression vector may include a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome.
  • Such vectors may be obtained commercially or be assembled from the sequences described by methods well known in the art.
  • Enhancers are cis-acting elements of DNA that act on a promoter to increase the level of transcription.
  • Vectors will also generally include origins of replication in addition to the selectable markers.
  • the polymerases of the present invention are selected according their ability to catalyse improved incorporation of nucleotides which have been modified at the 3' sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3' hydroxyl group, compared to a control polymerase.
  • nucleosides or nucleotides which are incorporated by the polymerases of the present invention comprise a purine or pyrimidine base and a ribose or deoxyribose sugar moiety which has a blocking group covalently attached thereto, preferably at the 3'O position, which renders the molecules useful in techniques requiring blocking of the 3'-OH group to prevent incorporation of additional nucleotides, such as for example in sequencing reactions, polynucleotide synthesis, nucleic acid amplification, nucleic acid hybridisation assays, single nucleotide polymorphism studies, and other such techniques.
  • Preferred modified nucleotides are exemplified in International Patent Application publication number WO 2004/018497 in the name of Solexa Limited, which reference is incorporated herein in its entirety.
  • the R' group of the modified nucleotide or nucleoside is an alkyl or substituted alkyl.
  • the -Z group of the modified nucleotide or nucleoside is of formula -C(R') 2 -N 3 .
  • the modified nucleotide or nucleoside includes a Z group which is an azido methyl group.
  • the preferred polymerases of the invention having the motif A region sequences YAV or YAS are particularly preferred for incorporation of nucleotide analogues wherein Z is an azido methyl group.
  • the modified nucleotide can be linked via the base to a detectable label by a desirable linker, which label may be a fluorophore, for example.
  • the detectable label may instead, if desirable, be incorporated into the blocking groups of formula "Z".
  • the linker can be acid labile, photolabile or contain a disulfide linkage.
  • Other linkages, in particular phosphine-cleavable azide-containing linkers, may be employed in the invention as described in greater detail in WO 2004/018497 , the contents of which are incorporated herein in their entirety.
  • the modified nucleotide or nucleoside will have a base attached to a detectable label via a cleavable linker, characterised in that the cleavable linker contains a moiety selected from the group comprising: (wherein X is selected from the group comprising O, S, NH and NQ wherein Q is a C 1-10 substituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH and N(allyl), T is hydrogen or a C 1-10 substituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside).
  • X is selected from the group comprising O, S, NH and NQ wherein Q is a C 1-10 substituted or unsubstituted alkyl group
  • Y is selected from the group comprising O, S, NH and N(allyl)
  • T is hydrogen or a C 1-10 substituted or unsubstit
  • the detectable abel comprises a fluorescent label.
  • Suitable fluorophores are well known in the art.
  • each different nucleotide type will carry a different fluorescent label. This facilitates the identification of incorporation of a particular nucleotide.
  • modified Adenine, Guanine, Cytosine and Thymine would all have attached a separate fluorophore to allow them to be discriminated from one another readily.
  • the inventors have found that the altered polymerases are capable of incorporating modified nucleotide analogues carrying a number of different fluorescent labels.
  • the polymerases are capable of incorporating all four bases.
  • mutant enzymes having the motif A region sequences YAV and YAS are the most preferred for incorporation of nucleotide analogues containing O -azido methyl functionality at the 3' position. It will be appreciated that for other nucleotide analogues the preferred amino acid sequence of the motif A region for optimum incorporation may vary. For any given nucleotide analogue, optimum region A sequence preferences may be determined by experiment, for example by construction of a library or discrete number of mutants followed by testing of individual variants in an incorporation assay system.
  • the preferred region A variant polymerases identified herein also exhibit improved incorporation of modified nucleotides containing a number of different O -amido methyl blocking groups bearing further substituents which increase the overall size of the 3' blocking group, also modified nucleotides containing 3' blocking groups that are not based on the O -azido methyl functionality and modified nucleotides bearing a number of different fluorescent labels.
  • the altered polymerases of the invention are capable of improved incorporation of a wide range of modified nucleotides having large 3' substituent groups of differing sizes and of varied chemical nature.
  • the invention relates to use of an altered polymerase according to the invention for the incorporation of a nucleotide into a polynucleotide.
  • the nucleotide is a modified nucleotide which has been modified at the 3' sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3' hydroxyl group.
  • the polymerases of the invention may be used in any area of technology where it is required/desirable to be able to incorporate modified nucleotides having a substituent at the 3' sugar hydroxyl position which is larger in size than the naturally occurring hydroxyl group into a polynucleotide chain. They may be used in any area of technology where any of the desirable properties of the enzyme, specifically improved rate of incorporation of nucleotide analogues, ability to incorporate labelled (e.g. fluorescent) analogues, ability to incorporate all four bases, activity at low temperature and/or over a wide temperature range, activity at low substrate concentration, activity over multiple cycles of incorporation, and high fidelity, provides an advantage. This could be a practical, technical or economic advantage.
  • the altered polymerases exhibit desirable properties in relation to incorporation of modified nucleotides having a large 3' substituent, the utility of the enzymes need not be confined to incorporation of such nucleotide analogues.
  • the desirable properties of the altered polymerase may provide advantages in relation to incorporation of any other nucleotide, including unmodified nucleotides, relative to enzymes known. in the art.
  • the altered polymerases of the invention may be used to incorporate any type of nucleotide that they have the ability to incorporate.
  • the polymerases of the present invention are useful in a variety of techniques requiring incorporation of a nucleotide into a polynucleotide, which include sequencing reactions, polynucleotide synthesis, nucleic acid amplification, nucleic acid hybridisation assays, single nucleotide polymorphism studies, and other such techniques. All such uses and methods utilizing the modified polymerases of the invention are included within the scope of the present invention.
  • the invention also relates to a method for incorporating nucleotides which have been modified at the 3' sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3' hydroxyl group into DNA comprising allowing the following components to interact:
  • the above components are allowed to interact under conditions which permit the formation of a phosphodiester linkage between the 5' phosphate group of a modified nucleotide and a free 3' hydroxyl group on the DNA template, whereby the modified nucleotide is incorporated into a polynucleotide.
  • the incorporation reactions may occur in free solution or the DNA templates may be fixed to a solid support.
  • polymerases of the invention In order for the polymerases of the invention to be of practical use in any area of technology where it is required/desirable to be able to incorporate modified nucleotides having a substituent at the 3' sugar hydroxyl position which is larger in size than the naturally occurring hydroxyl group into a polynucleotide chain, it is required only that the enzyme be able to incorporate the analogue at an acceptable rate.
  • An "acceptable" rate of incorporation in practical terms may be defined as detectable (i.e.
  • the polymerase may exhibit a time to 50% product conversion on the second cycle of enzymology at 45°C, 50mM Tris, pH8, 4mM MgSO 4 , 0.05 (v/v) Tween, 50 ⁇ g/ml enzyme, and 2 ⁇ M 3' modified nucleotide of 19.5 min or less, more preferably 5 min or less and most preferably 1.5 min or less.
  • the rate of incorporation of the 3' modified nucleotide analogue exhibited by a mutant enzyme may be similar to the rate of incorporation of unmodified nucleotides exhibited by the mutant enzyme, or the equivalent wild type enzyme from which it is derived. However, it is not necessary for the rate of incorporation of 3' modified analogues to be similar to that of unmodified nucleotides for a mutant enzyme to be of practical use. All that is required is for the incorporation rate of the 3' modified analogue to be "acceptable" according to the definition provided above on at least one cycle of incorporation.
  • the rate of incorporation may be less than, equal to or greater than the rate of incorporation of unmodified nucleotides and/or nucleotides modified such that the substituent is smaller than a hydroxyl group.
  • the altered polymerases of the invention may be used to incorporate modified nucleotides into a polynucleotide chain in the context of a sequencing-by-synthesis protocol.
  • the nucleotides which have been modified at the 3' sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3' hydroxyl group will be detected in order to determine the sequence of a DNA template.
  • the invention provides a method of sequencing DNA comprising allowing the following components to interact:
  • the DNA template for a sequencing reaction will typically comprise a double-stranded region having a free 3' hydroxyl group which serves as a primer or initiation point for the addition of further nucleotides in the sequencing reaction.
  • the region of the DNA template to be sequenced will overhang this free 3' hydroxyl group on the complementary strand.
  • the primer bearing the free 3' hydroxyl group may be added as a separate component (e.g. a short oligonucleotide) which hybridises to a region of the template to be sequenced.
  • the primer and the template strand to be sequenced may each form part of a partially self-complementary nucleic acid strand capable of forming an intramolecular duplex, such as for example a hairpin loop structure.
  • Nucleotides are added successively to the free 3' hydroxyl group, resulting in synthesis of a polynucleotide chain in the 5' to 3' direction. After each nucleotide addition the nature of the base which has been added will be determined, thus providing sequence information for the DNA template.
  • modified nucleotides can act as chain terminators. Once the modified nucleotide has been incorporated into the growing polynucleotide chain complementary to the region of the template being sequenced there is no free 3'-OH group available to direct further sequence extension and therefore the polymerase can not add further nucleotides. Once the nature of the base incorporated into the growing chain has been determined, the 3' block may be removed to allow addition of the next successive nucleotide. By ordering the products derived using these modified nucleotides it is possible to deduce the DNA sequence of the DNA template.
  • Such reactions can be done in a single experiment if each of the modified nucleotides has attached a different label, known to correspond to the particular base, to facilitate discrimination between the bases added at each incorporation step.
  • a separate reaction may be carried out containing each of the modified nucleotides separately.
  • the modified nucleotides carry a label to facilitate their detection.
  • this is a fluorescent label.
  • Each nucleotide type may carry a different fluorescent label.
  • the detectable label need not be a fluorescent label. Any label can be used which allows the detection of the incorporation of the nucleotide into the DNA sequence.
  • One method for detecting the fluorescently labelled nucleotides comprises using laser light of a wavelength specific for the labelled nucleotides, or the use of other suitable sources of illumination.
  • the fluorescence from the label on the nucleotide may be detected by a CCD camera.
  • the DNA templates are immobilised on a surface they may preferably be immobilised on a surface to form a high density array.
  • the high density array comprises a single molecule array, wherein there is a single DNA molecule at each discrete site that is detectable on the array.
  • Single-molecule arrays comprised of nucleic acid molecules that are individually resolvable by optical means and the use of such arrays in sequencing are described, for example, in WO 00/06770 , the contents of which are incorporated herein by reference.
  • Single molecule arrays comprised of individually resolvable nucleic acid molecules including a hairpin loop structure are described in WO 01/57248 , the contents of which are also incorporated herein by reference.
  • the polymerases of the invention are suitable for use in conjunction with single molecule arrays prepared according to the disclosures of WO 00/06770 of WO 01/57248 .
  • the scope of the invention is not intended to be limited to the use of the polymerases in connection with single molecule arrays.
  • Single molecule array-based sequencing methods may work by adding fluorescently labelled modified nucleotides and an altered polymerase to the single molecule array.
  • Complementary nucleotides would base-pair to the first base of each nucleotide fragment and would be added to the primer in a reaction catalysed by the improved polymerase enzyme. Remaining free nucleotides would be removed.
  • the identity of the incorporated modified nucleotide would reveal the identity of the base in the sample sequence to which it is paired.
  • the cycle of incorporation, detection and identification would then be repeated approximately 25 times to determine the first 25 bases in each oligonucleotide fragment attached to the array, which is detectable.
  • the first 25 bases for the hundreds of millions of oligonucleotide fragments attached in single copy to the array could be determined.
  • the invention is not limited to sequencing 25 bases. Many more or less bases could be sequenced depending on the level of detail of sequence information required and the complexity of the array.
  • the generated sequences could be aligned and compared to specific reference sequences. This would allow determination of any number of known and unknown genetic variations such as single nucleotide polymorphisms (SNPs) for example.
  • SNPs single nucleotide polymorphisms
  • the utility of the altered polymerases of the invention is not limited to sequencing applications using single-molecule arrays.
  • the polymerases may be used in conjunction with any type of array-based (and particularly any high density array-based) sequencing technology requiring the use of a polymerase to incorporate nucleotides into a polynucleotide chain, and in particular any array-based sequencing technology which relies on the incorporation of modified nucleotides having large 3' substituents (larger than natural hydroxyl group), such as 3' blocking groups.
  • the polymerases of the invention may be used for nucleic acid sequencing on essentially any type of array formed by immobilisation of nucleic acid molecules on a solid support.
  • suitable arrays may include, for example, multi-polynucleotide or clustered arrays in which distinct regions on the array comprise multiple copies of one individual polynucleotide molecule or even multiple copies of a small-number of different polynucleotide molecules (e.g. multiple copies of two complementary nucleic acid strands).
  • polymerases of the invention may be utilised in the nucleic acid sequencing method described in WO 98/44152 , the contents of which are incorporated herein by reference.
  • This International application describes a method of parallel sequencing of multiple templates located at distinct locations on a solid support. The method relies on incorporation of labelled nucleotides into a polynucleotide chain.
  • the polymerases of the invention may be used in the method described in International Application WO 00/18957 , the contents of which are incorporated herein by reference.
  • This application describes a method of solid-phase nucleic acid amplification and sequencing in which a large number of distinct nucleic acid molecules are arrayed and amplified simultaneously at high density via formation of nucleic acid colonies and the nucleic acid colonies are subsequently sequenced.
  • the altered polymerases of the invention may be utilised in the sequencing step of this method.
  • Multi-polynucleotide or clustered arrays of nucleic acid molecules may be produced using techniques generally known in the art.
  • WO 98/44151 and WO 00/18957 both describe methods of nucleic acid amplification which allow amplification products to be immobilised on a solid support in order to form arrays comprised of clusters or "colonies" of immobilised nucleic acid molecules.
  • the contents of WO 98/44151 and WO 00/18957 relating to the preparation of clustered arrays and use of such arrays as templates for nucleic acid sequencing are incorporated herein by reference.
  • the nucleic acid molecules present on the clustered arrays prepared according to these methods are suitable templates for sequencing using the polymerases of the invention.
  • the invention is not intended to use of the polymerases in sequencing reactions carried out on clustered arrays prepared according to these specific methods.
  • the polymerases of the invention may further be used in methods of fluorescent in situ sequencing, such as that described by Mitra et al. Analytical Biochemistry 320, 55-65, 2003 .
  • the variant polymerases have increased activity towards the modified nucleotides utilised in the experiments. Thus, they appear to incorporate nucleotide analogues which have been modified at the 3' sugar hydroxyl such that the substituent is larger in size than the naturally occurring 3' hydroxyl group in primer extension assays faster than previously available polymerases which can act as a control polymerase, such as the 9°N (-exo)Y409V/A485L double mutant (designated 9DM herein).
  • 9DM 9°N (-exo)Y409V/A485L double mutant
  • a sequenced and functionally validated clone of the polymerase is used as the starting material.
  • the plasmid from the 9°N (-exo) Y409V/A485L (9°N DM) double mutant was utilised (SEQ ID N0:15) .
  • Table III shows the nucleotide sequences of the oligonucleotide primers used in order to generate the library of mutant 9°N DNA polymerases in the mutagenesis experiments.
  • Long name Short name Nucleotide sequence reamp 9°N atg long 133-2 SEQ ID NO: 1 CACTCATGATTAGATCTCGTGCAGC 9°N atg long 133-1 SEQ ID NO: 2 9°N 1221-1198 125-1 SEQ ID NO: 3 CGAGCGGAAGTCTAAATACACAAT 9°N 1198-1221 119-5 SEQ ID NO: 4 3ATTGTGTATTTAGACTTCCGCTCG 9°N 1198-1251 NNK 119-3 SEQ ID NO: 5 9°N 1483-1428 119-6 SEQ ID NO: 6 9°N 1483-1460 119-7 SEQ ID NO: 7 GTAGAAGCTGTTGGCGAGGATCTT 9°N 1460-1483 125-2 SEQ ID NO: 8 AAGATCCTCGCCAACAGCTTCTAC 9°N term
  • a portion of the ligation was transformed directly into an strain of E. coli, such as BL21 (DE3) plysS, that allows expression of the variant genes, or alternatively into an intermediate host, such as E. coli DH5Alpha.
  • Dilute anti-DIG alkaline phosphatase-conjugated antibody (Roche) 1:5000 in 10 ml DIG blocking buffer per small round filter (50 ml for big 22 cm 2 plates). Incubate filters with antibody for 45 min at room temperature on rocker. Wash filters twice with DIG wash buffer for 15 min each on rocker. Equilibrate filters with 10 ml DIG detection buffer (Roche) for 2 min on rocker (50 ml for big 22 cm 2 plates). Add 20 ⁇ l NBT/BCIP (Roche) to 10 ml fresh DIG detection solution per small round filter and incubate at room temperature on rocker for 20 min (100 ⁇ l NBT/BCIP to 50 ml DIG detection solution for big 22 cm 2 plates. Petri dishes containing filters should be wrapped in foil during detection. Rinse filter thoroughly with tap water for a few minutes to stop reaction. Dry filters on 3MM paper at 37°C.
  • region C amino acids 493, 494 and 496 of 9°N polymerase
  • the polymerase variant ED is a derivative of the 9°N DM DNA polymerase that bears the following mutations: L408V; V409A; P410L.
  • This clone can be constructed in a variety of ways following standard molecular biology methods. One method for the construction of a gene encoding ED is described herein, and was used in our experiments.
  • DNA encoding the 2E gene product (as described in Example 1) was isolated and digested with restriction enzyme XhoI (New England Biolabs, NEB) following manufacturer's instructions. The resulting DNA fragment that encodes the region spanning amino acids 408-410 was purified from an agarose gel using standard methods. This DNA fragment was then ligated into the XhoI-digested vector backbone of the 9°N DM plasmid pNEB917(NEB; as described Example 1) using T4 DNA ligase (NEB), following standard procedures. The background of religated vector backbone was reduced by prior treatment with calf-intestinal phosphatase (NEB) following standard procedures. The product of the ligation reaction was transformed into library efficiency E .
  • restriction enzyme XhoI New England Biolabs, NEB
  • coli DH5alpha competent cells (Invitrogen) following manufacturers instructions. Colonies were then screened by PCR using oligonucleotide primers that bind specifically to the 2E sequence (primer 2E 408-10; sequence 5' GACTTCCGCTCGGTTGCGTTG 3' SEQ ID NO:23) and the 9°N DM vector backbone (primer sequence 5' AAGCCCCTCACGTAGAAGCC 3' SEQ ID NO:24) in a 20 ⁇ l reaction mixture of the following composition: DNA/colony solution in water, 1 ⁇ l; 0.1 ⁇ M primers; 200uM dNTPs; 1unit Taq DNA polymerase; 2 ⁇ l 10x Taq reaction buffer. Reactions were cycled 30 times at 94°C, 1min, 55°C, 1min, 72°C, 2min and positive colonies identified by inspection of the reaction products by agarose gel electrophoresis.
  • the construction of a library of mutant proteins can be performed by different methods.
  • the method used in this work involved two stages: the formation of a degenerate mix of DNA templates that encode the mutant polymerases, and the cloning of these DNA fragments into a suitable expression vector.
  • the pool of degenerate DNA templates was formed by PCR using oligonucleotide primers Xba 408-410 (5' GTGTATCTAGACTTCCGCTCGNNKNNKNNKTCAATCATCATAACCCACAAC 3' SEQ ID NO:25; N, equimolar mixture of G, A, T and C bases; K, an equimolar mixture of G and T bases) and BamHI 3' 9°N
  • the DNA fragment was amplified by PCR in a 20 ⁇ l reaction as described above, using 10ng 9°N DM template DNA.
  • the 1200bp DNA product was gel purified using a gel purification method (Qiagen) following the manufacturer's instructions, and the fragment digested with restriction enzymes XbaI and BamHI (NEB), using the 10x BamHI buffer (NEB) as recommended by the manufacturer.
  • these DNA fragments were cloned into the XbaI-BamHI vector backbone of expression plasmid pSV13, a derivative of pNEB917 (NEB), which was constructed in a two step procedure, as follows. Firstly, the XbaI restriction site in the polylinker of the pNEB917 plasmid was destroyed by cutting the plasmid with XbaI, extension of the resulting 3' overhang with Klenow polymerase, and relegation of blunt-ended DNA with T4 ligase, using standard molecular biology procedures (Sambrook and Russell, 2001).
  • This plasmid, pSV12 was then mutated further to form pSV13 by the introduction of a unique XbaI site at position 1206 in the 9°N DNA sequence.
  • This mutation was performed by PCR mutagenesis using oligonucleotide primers XbaI mut 5' (5' GGGACAACATTGTGTATCTAGACTTCCGCTCGCTGGTGCCTTC 3' SEQ ID NO:27) and 1769R (5' GTCTATCACAGCGTACTTCTTCTTCG 3' SEQ ID NO:28) in one reaction, and XbaI mut 3' (5' GAAGGCACCAGCGAGCGGAAGTCTAGATACACAATGTTGTCCC 3' SEQ ID NO:29) and 1032F 5' GGCCAGAGCCTCTGGGACGTC 3' SEQ ID NO:30) in a second reaction.
  • PCR cycling conditions for these amplifications were 30 cycles of 95°C for 1 min, 60°C for 30sec and 72°C for 1.5 min using 1.5 units of Taq and 1 ⁇ M of primers per reaction.
  • the products of these PCRs were two partially overlapping DNA fragments that were then used in a second PCR reaction with primers 1032F/1769R to produce a 737bp DNA product, using identical reaction conditions.
  • This final product was digested with XhoI (XhoI sites at 1040 and 1372 flanked the XbaI mutation) and ligated with XhoI cut pSV12. The orientation of the insertion was checked by restriction digestion and the resulting plasmid named pSV13. Both pSV12 and pSV13 were sequenced over the regions where the changes were made.
  • the method used to make this degenerate pool of DNA fragments involved a 3-way PCR reaction with primers Xba 408-410 (described above) and 493-6Rev (5' GCGTAGCCGTAMNNGCCMNNMNNGCTGTTGGCGAGGATTTTGATCAGCCTC SEQ ID NO:31; M represents an equimolar mixture of bases A and C, and N represents an equimolar mixture of all 4 bases) in the first reaction, using reaction conditions as described above.
  • the product of this reaction was then used as the template DNA in a second PCR reaction with primers Xba 408-410 and BamHI 3' (5' GCGCGCGGATCCTCACTTCTTCCCCTTCACC SEQ ID NO:32) using conditions described above.
  • the DNA product from this second PCR was digested with restriction enzymes XbaI and BamHI and cloned into vector pSV13.
  • Example 1 The screening procedure that was used is described in Example 1. In one experiment, a total of 251 positive colonies were identified from a library of 4.2 x 10 4 colonies (A and C regions randomised simultaneously), and in a second experiment, 255 positive colonies were identified from a library of 7 x 10 4 colonies (region A randomised alone).
  • the incorporation reactions were set up as follows: 10nM biotinylated substrate DNA was added to the wells of a Strepavidin-coated microtitre plate (Sigma) in 50mM Tris-HCl, pH 8. Wells were washed with three times with PBS buffer containing 0.05% Tween-20 (PBS-T) before the addition of 0.2 ⁇ M ffT-DIG in a volume of 40 ⁇ L buffer (50mM Tris HCl, pH8, 0.05% Tween-20, 4mM MgSO 4 ) and 10 ⁇ L bacterial supernatant containing the recombinant enzyme (above).
  • Reactions were incubated for 15 mins at 30°C or 65°C and stopped by the addition of 10 ⁇ L 0.5M EDTA. The reaction solutions were removed and the wells were washed with PBS-T as before. The presence of incorporated DIG was detected using a horseradish peroxidase-linked anti-DIG antibody (Roche) and 3,3',5,5'-tetramethylbenzidine substrate (Sigma).
  • the recombinant proteins were purified from the bacterial supernatants and the quantity of recombinant protein determined. It was then possible to compare the rates of incorporation reactions of different polymerase enzymes by mixing the DNA substrate, enzyme and ffT-DIG in solutions and stopping the reactions at different times by diluting an aliquot (50 ⁇ L) of the reaction mixture into 10 ⁇ L 0.5M EDTA solution. These different samples were then added to the wells of a streptavidin-coated microtitre plate. The amount of DIG incorporation was measured in an identical fashion to the above procedure.
  • the DNA encoding the mutant DNA polymerases was transformed into BL21(DE3) RIL Codon Plus competent cells (Stratagene) using the manufacturer's instructions. Single colonies of transformed bacteria were used to inoculate a flask of LB broth containing carbenicillin and chloramphenicol and this culture was grown with shaking overnight at 37°C. This culture was used to inoculate a larger volume of LB broth containing carbenicillin and chloramphenicol at a 1:100 dilution, and this culture was then grown with shaking at 37°C to an optical density of approximately 0.4-0.6 at 600nm. Protein expression was induced by the addition of 1mM IPTG, and the culture grown for a further 2.5h at 37°C.
  • the samples were then heated at 75°C for 30 minutes and centrifuged at 18000rpm for 30 mins at 4°C to remove denatured protein. After washing the pellet in wash buffer (above), the samples were diluted 3-fold in 10mM Tris/HCl, pH 7.5, 300mM NaCL, 0.1mM EDTA, 0.05% Triton-X-100 and applied to a DEAE FF HiTrap chromatography column (Amersham Biosciences). The target protein was located in the flow through fraction, and further purification is achieved by chromatography using a Heparin FF' column (Amersham Biosciences), after dilution in 10mM KPO4, pH 6.9, 0.1mM EDTA, 0.05% Triton-X-100. Fractions were eluted from the column in the same buffer and those containing the target protein were identified by SDS-PAGE and staining with Coommassie Brilliant Blue staining as described in Sambrook and Russell, 2001.
  • the mutant polymerases were assessed for their ability to incorporate a 3' blocked nucleotide analogue using a gel-based assay as described in generic form in Example 1.
  • the rates of incorporation of the modified nucleotides were compared to purified forms of related DNA polymerases in both a single round of incorporation and through two rounds of nucleotide incorporation.
  • the DNA template that was used for these experiments was a commercially-synthesised biotinylated oligonucleotide.
  • the method for two cycles of incorporation involves the binding of the DNA primer-template to streptavidin-coated beads (Dynalbeads). These are prepared as follows:
  • the beads must be agitated every 3-5 minutes to ensure that the reactions are able to proceed.
  • the first cycle of enzymatic incorporation is performed on these beads as follows:
  • reactions are incubated at 45 °C for 15 minutes
  • a 5 ⁇ L sample is removed and added to gel loading buffer with EDTA
  • a second 5 ⁇ L sample is also removed to which is added 1 ⁇ L of 4 natural nucleotide triphosphates (Sigma) and incubated for a further 10 minutes at 45°C.
  • To 5 ⁇ L of this sample is added 1 ⁇ l gel loading buffer with EDTA
  • TCEP tri(carboxyethyl) phosphine
  • incorporation mix 2 After removal of the TE buffer, a second cycle of enzymatic incorporation is then performed by the addition of incorporation mix 2, as follows: To the deblocked beads (above) add: 1 ⁇ L 0.1 mM 3' modified nucleotide analogue 5 ⁇ L 500 mM Tris pH 8.0 2.5 ⁇ L 1% Tween-20 2 ⁇ L 100 mM MgSO4 50 ⁇ g/ml purified DNA polymerase H 2 O to make up the volume to 50 ⁇ L
  • sequences of amino acids 408-410 for DNA polymerases exhibiting improved activity compared to 9°N DM YST FAI FAP VAP AAA YAS & YAV & YGI & YSG & SGG # CST IAL CGG SAL SAA CAA YAA QAS VSS VAG VAV FAV AGI YSS AAT FSS VAL # this polymerase also contained the F493H mutation in region C & this clone was obtained from a library in which both regions A and C were randomised; however, the amino acid sequence of region C was identical to wild type
  • the 9°N ED polymerase variant was capable of greater than 90% substrate conversion at a 2 ⁇ M concentration of nucleotide within 3 minutes at 45 °C
  • point mutations were introduced into the 9°N DM polymerase to change codon 409 into Alanine (9°N DM V409A) or Glycine (9°N DM V409G).
  • Example 2 To measure the incorporation of 3' blocked nucleotide analogues at different temperatures, a modified version of the microtitre-based incorporation assay (described in Example 2) was developed.
  • a mixture of biotinylated DNA substrate at 10nM concentration and purified polymerase enzyme at 50nM concentration was mixed in a buffer comprising 50mM Tris, 0.05% Tween 20 and 10mM KCl.
  • a mixture of 0.2 ⁇ M ffT-DIG (as described in Example 2) and 4mM MgSO4 was made in an identical buffer to that described for the first mixture. Reaction volumes were adjusted so that the mixture in tube 1 comprised 40 ⁇ L, and in tube 2, 10 ⁇ L.
  • reaction volumes were scaled appropriately. All tubes were prewarmed at the appropriate temperature and the contents of tube 2 was added to tube 1 to initiate the incorporation reaction. At various time intervals, 50 ⁇ L of the combined reaction mixture was removed and the incorporation reaction terminated by the addition of 100 mM EDTA (final concentration). The different aliquots of stopped reaction mixture were then added to separate wells of a streptavidin-coated microtitre plate, and the extent of ffT-DIG incorporation was assessed as described in Example 2.
  • Example 2 To determine over what range of nucleotide concentrations the modified DNA polymerases were capable of efficient incorporation of the 3' modified nucleotide analogues, a two cycle incorporation experiment was performed as described in Example 2. The incorporation rates for a 3' O-azido methyl-modified nucleotide (described in Example 2) were measured over a range of nucleotide concentrations from 0.2 ⁇ M to 50 ⁇ M of ffT, and the results for the 9oN YAV variant polymerase (exo-) are shown in figure 6 .

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Saccharide Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
EP10180445A 2003-09-11 2004-09-10 ADN polymérases modifiées pour une meilleure intégration d'analogues de nucléotides Revoked EP2325304B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB0321306.3A GB0321306D0 (en) 2003-09-11 2003-09-11 Modified polymerases for improved incorporation of nucleotide analogues
EP04768438A EP1664287B1 (fr) 2003-09-11 2004-09-10 Adn polymerases de type b modifies pour l'incorporation amelioree d'analogues nucleotidiques

Related Parent Applications (2)

Application Number Title Priority Date Filing Date
WOPCT/GB2004/003891 Previously-Filed-Application 2004-09-10
EP04768438.6 Division 2004-09-10

Publications (2)

Publication Number Publication Date
EP2325304A1 true EP2325304A1 (fr) 2011-05-25
EP2325304B1 EP2325304B1 (fr) 2012-05-09

Family

ID=29226911

Family Applications (2)

Application Number Title Priority Date Filing Date
EP04768438A Expired - Lifetime EP1664287B1 (fr) 2003-09-11 2004-09-10 Adn polymerases de type b modifies pour l'incorporation amelioree d'analogues nucleotidiques
EP10180445A Revoked EP2325304B1 (fr) 2003-09-11 2004-09-10 ADN polymérases modifiées pour une meilleure intégration d'analogues de nucléotides

Family Applications Before (1)

Application Number Title Priority Date Filing Date
EP04768438A Expired - Lifetime EP1664287B1 (fr) 2003-09-11 2004-09-10 Adn polymerases de type b modifies pour l'incorporation amelioree d'analogues nucleotidiques

Country Status (8)

Country Link
US (7) US8460910B2 (fr)
EP (2) EP1664287B1 (fr)
JP (1) JP5706057B2 (fr)
AT (2) ATE532856T1 (fr)
DK (1) DK2325304T3 (fr)
GB (1) GB0321306D0 (fr)
HK (1) HK1156353A1 (fr)
WO (1) WO2005024010A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9605310B2 (en) 2001-12-04 2017-03-28 Illumina Cambridge Limited Labelled nucleotides
EP3002289B1 (fr) 2002-08-23 2018-02-28 Illumina Cambridge Limited Nucleotides modifies pour le sequençage de polynucleotide
CN107922929A (zh) * 2015-09-09 2018-04-17 凯杰有限公司 聚合酶
US10407721B2 (en) 2013-03-15 2019-09-10 Illumina Cambridge Limited Modified nucleosides or nucleotides
US10487102B2 (en) 2002-08-23 2019-11-26 Illumina Cambridge Limited Labelled nucleotides
US10995111B2 (en) 2003-08-22 2021-05-04 Illumina Cambridge Limited Labelled nucleotides
WO2023020728A1 (fr) * 2021-08-14 2023-02-23 Illumina, Inc. Polymérases, compositions et procédés d'utilisation
US12104182B2 (en) 2018-12-05 2024-10-01 Illumina, Inc. Polymerases, compositions, and methods of use

Families Citing this family (399)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1790736A3 (fr) 2000-10-06 2007-08-15 The Trustees Of Columbia University In The City Of New York Procédé massivement parallèle pour décoder l'adn et l'arn
US9708358B2 (en) 2000-10-06 2017-07-18 The Trustees Of Columbia University In The City Of New York Massive parallel method for decoding DNA and RNA
US7057026B2 (en) 2001-12-04 2006-06-06 Solexa Limited Labelled nucleotides
GB0321306D0 (en) 2003-09-11 2003-10-15 Solexa Ltd Modified polymerases for improved incorporation of nucleotide analogues
US7622279B2 (en) * 2004-03-03 2009-11-24 The Trustees Of Columbia University In The City Of New York Photocleavable fluorescent nucleotides for DNA sequencing on chip constructed by site-specific coupling chemistry
US20070048748A1 (en) * 2004-09-24 2007-03-01 Li-Cor, Inc. Mutant polymerases for sequencing and genotyping
US8623628B2 (en) 2005-05-10 2014-01-07 Illumina, Inc. Polymerases
GB0509508D0 (en) * 2005-05-10 2005-06-15 Solexa Ltd Improved polymerases
WO2007002204A2 (fr) * 2005-06-21 2007-01-04 The Trustees Of Columbia University In The City Of New York Procedes de pyrosequencage et compositions associees
US7982029B2 (en) 2005-10-31 2011-07-19 The Trustees Of Columbia University In The City Of New York Synthesis of four color 3′O-allyl, modified photocleavable fluorescent nucleotides and related methods
US8796432B2 (en) 2005-10-31 2014-08-05 The Trustees Of Columbia University In The City Of New York Chemically cleavable 3'-o-allyl-DNTP-allyl-fluorophore fluorescent nucleotide analogues and related methods
EP1957983A4 (fr) * 2005-11-21 2010-03-24 Univ Columbia Immunocapture numérique multiplex utilisant une bibliothèque de marqueurs de masse photoclivable
CN101460953B (zh) 2006-03-31 2012-05-30 索雷克萨公司 用于合成分析的序列的系统和装置
EP2074211B1 (fr) * 2006-09-06 2013-03-13 Medical Research Council Polymérases d'ADN incorporant des analogues de nucléotides marqués au colorant
DK2076592T3 (en) 2006-10-23 2017-07-24 Medical Res Council POLYMERASE
KR100777230B1 (ko) * 2006-11-30 2007-11-28 한국해양연구원 써모코커스 유래 돌연변이 ｄｎａ 중합효소들 및 그의유전자들
DE112007002932B4 (de) 2006-12-01 2015-08-06 The Trustees Of Columbia University In The City Of New York Vierfarben DNA-Sequenzierung mittels Synthese unter Verwendung von abspaltbaren, reversiblen, fluoreszierenden Nucleotidterminatoren
US11940413B2 (en) 2007-02-05 2024-03-26 IsoPlexis Corporation Methods and devices for sequencing nucleic acids in smaller batches
US11035823B2 (en) 2009-03-17 2021-06-15 Qiagen Sciences, Llc Methods and devices for sequencing nucleic acids in smaller batches
US8481259B2 (en) 2007-02-05 2013-07-09 Intelligent Bio-Systems, Inc. Methods and devices for sequencing nucleic acids in smaller batches
US20110014611A1 (en) 2007-10-19 2011-01-20 Jingyue Ju Design and synthesis of cleavable fluorescent nucleotides as reversible terminators for dna sequences by synthesis
EP2209911B1 (fr) 2007-10-19 2013-10-16 The Trustees of Columbia University in the City of New York Séquençage d'adn avec des terminateurs réversibles nucléotidiques non fluorescents et des terminateurs nucléotidiques modifiés par un marqueur séparable et un composé de déoxyinosine contenant un terminateur réversible
US8617811B2 (en) 2008-01-28 2013-12-31 Complete Genomics, Inc. Methods and compositions for efficient base calling in sequencing reactions
US9017973B2 (en) 2008-03-19 2015-04-28 Intelligent Biosystems, Inc. Methods and compositions for incorporating nucleotides
US8883999B2 (en) 2008-03-19 2014-11-11 Intelligent Bio Systems, Inc. Methods and solutions for inhibiting undesired cleaving of labels
WO2009131919A2 (fr) * 2008-04-22 2009-10-29 New England Biolabs, Inc. Polymérases pour incorporer des nucléotides modifiés
US8039817B2 (en) 2008-05-05 2011-10-18 Illumina, Inc. Compensator for multiple surface imaging
EP2294214A2 (fr) * 2008-05-07 2011-03-16 Illumina, Inc. Compositions et procédés pour fournir des substances à une matrice et provenant de celle-ci
CA2742298C (fr) 2008-11-03 2019-09-10 The Regents Of The University Of California Procedes de detection d'acides nucleiques resistant a une modification
US20100151473A1 (en) * 2008-12-10 2010-06-17 Yeakley Joanne M Methods and compositions for hybridizing nucleic acids
US8236532B2 (en) 2008-12-23 2012-08-07 Illumina, Inc. Multibase delivery for long reads in sequencing by synthesis protocols
WO2010091400A2 (fr) 2009-02-09 2010-08-12 Frederic Zenhausern Améliorations se rapportant à une analyse
EP2427572B1 (fr) * 2009-05-01 2013-08-28 Illumina, Inc. Procédés de séquençage
US20120202276A1 (en) 2010-02-26 2012-08-09 Life Technologies Corporation Modified Proteins and Methods of Making and Using Same
EP2539471B1 (fr) * 2010-02-26 2014-08-06 Life Technologies Corporation Procédés de séquencage utilisant une polymérase modifiée
US8951940B2 (en) 2010-04-01 2015-02-10 Illumina, Inc. Solid-phase clonal amplification and related methods
PL2556171T3 (pl) 2010-04-05 2016-04-29 Prognosys Biosciences Inc Oznaczenia biologiczne kodowane przestrzennie
US10787701B2 (en) 2010-04-05 2020-09-29 Prognosys Biosciences, Inc. Spatially encoded biological assays
US20190300945A1 (en) 2010-04-05 2019-10-03 Prognosys Biosciences, Inc. Spatially Encoded Biological Assays
GB201007384D0 (en) * 2010-04-30 2010-06-16 Medical Res Council Enzymes
US9051263B2 (en) 2010-08-25 2015-06-09 Pacific Biosciences Of California, Inc. Functionalized cyanine dyes (PEG)
US9029103B2 (en) 2010-08-27 2015-05-12 Illumina Cambridge Limited Methods for sequencing polynucleotides
US8483969B2 (en) 2010-09-17 2013-07-09 Illuminia, Inc. Variation analysis for multiple templates on a solid support
WO2012050920A1 (fr) 2010-09-29 2012-04-19 Illumina, Inc. Compositions et procédés de séquençage d'acides nucléiques
US8753816B2 (en) 2010-10-26 2014-06-17 Illumina, Inc. Sequencing methods
WO2012058096A1 (fr) 2010-10-27 2012-05-03 Illumina, Inc. Microdispositifs et cartouches de biocapteurs pour analyse biologique ou chimique et systèmes et procédés associés
US8575071B2 (en) 2010-11-03 2013-11-05 Illumina, Inc. Reducing adapter dimer formation
WO2012061832A1 (fr) 2010-11-05 2012-05-10 Illumina, Inc. Liaison entre des lectures de séquences à l'aide de codes marqueurs appariés
WO2012074855A2 (fr) 2010-11-22 2012-06-07 The Regents Of The University Of California Procédés d'identification d'un transcrit cellulaire naissant d'arn
GB201106254D0 (en) 2011-04-13 2011-05-25 Frisen Jonas Method and product
WO2013049135A1 (fr) 2011-09-26 2013-04-04 Gen-Probe Incorporated Algorithmes de détermination de séquences
US10378051B2 (en) 2011-09-29 2019-08-13 Illumina Cambridge Limited Continuous extension and deblocking in reactions for nucleic acids synthesis and sequencing
EP2776165A2 (fr) 2011-11-07 2014-09-17 Illumina, Inc. Appareils de séquençage intégré et procédés d'utilisation
EP2788499B1 (fr) 2011-12-09 2016-01-13 Illumina, Inc. Base de numération étendue pour étiquettes polymères
WO2013117595A2 (fr) 2012-02-07 2013-08-15 Illumina Cambridge Limited Enrichissement et amplification ciblés d'acides nucléiques sur un support
NO2694769T3 (fr) 2012-03-06 2018-03-03
US9444880B2 (en) 2012-04-11 2016-09-13 Illumina, Inc. Cloud computing environment for biological data
US9315864B2 (en) 2012-05-18 2016-04-19 Pacific Biosciences Of California, Inc. Heteroarylcyanine dyes with sulfonic acid substituents
US10458915B2 (en) 2012-05-18 2019-10-29 Pacific Biosciences Of California, Inc. Heteroarylcyanine dyes
EP2870264A4 (fr) 2012-07-03 2016-03-02 Sloan Kettering Inst Cancer Évaluation quantitative de la reconstitution du répertoire des cellules t chez l'homme après une greffe allogénique de cellules souches hématopoïétiques
NL2017959B1 (en) 2016-12-08 2018-06-19 Illumina Inc Cartridge assembly
JP6478444B2 (ja) * 2012-09-28 2019-03-06 東洋紡株式会社 改変された耐熱性ｄｎａポリメラーゼ
CA2886974C (fr) 2012-10-17 2021-06-29 Spatial Transcriptomics Ab Procedes et produit d'optimisation de la detection localisee ou spatiale de l'expression genique dans un echantillon de tissu
US9116139B2 (en) 2012-11-05 2015-08-25 Illumina, Inc. Sequence scheduling and sample distribution techniques
US20160138063A1 (en) * 2013-01-04 2016-05-19 John Chaput Methds and compositions for replication of threose nucleic acids
US9805407B2 (en) 2013-01-25 2017-10-31 Illumina, Inc. Methods and systems for using a cloud computing environment to configure and sell a biological sample preparation cartridge and share related data
US9650406B2 (en) 2013-02-28 2017-05-16 Centrillion Technology Holdings Corporation Reversible terminator molecules and methods of their use
DK2964624T3 (en) 2013-03-08 2017-04-03 Illumina Cambridge Ltd RHODAMINE COMPOUNDS AND USE AS FLUORESCING LABELS
ES2620423T3 (es) 2013-03-08 2017-06-28 Illumina Cambridge Limited Compuestos de polimetina y su uso como marcadores fluorescentes
ES2724824T3 (es) 2013-03-13 2019-09-16 Illumina Inc Métodos para la secuenciación de ácidos nucleicos
US9146248B2 (en) 2013-03-14 2015-09-29 Intelligent Bio-Systems, Inc. Apparatus and methods for purging flow cells in nucleic acid sequencing instruments
AU2013382024B2 (en) 2013-03-14 2019-01-31 Illumina, Inc. Modified polymerases for improved incorporation of nucleotide analogues
WO2014144883A1 (fr) 2013-03-15 2014-09-18 The Trustees Of Columbia University In The City Of New York Molecules marquees par des amas de raman destinees a l'imagerie biologique
US9591268B2 (en) 2013-03-15 2017-03-07 Qiagen Waltham, Inc. Flow cell alignment methods and systems
US9771613B2 (en) 2013-04-02 2017-09-26 Molecular Assemblies, Inc. Methods and apparatus for synthesizing nucleic acid
US9279149B2 (en) 2013-04-02 2016-03-08 Molecular Assemblies, Inc. Methods and apparatus for synthesizing nucleic acids
US8808989B1 (en) 2013-04-02 2014-08-19 Molecular Assemblies, Inc. Methods and apparatus for synthesizing nucleic acids
US11384377B2 (en) 2013-04-02 2022-07-12 Molecular Assemblies, Inc. Reusable initiators for synthesizing nucleic acids
US11331643B2 (en) 2013-04-02 2022-05-17 Molecular Assemblies, Inc. Reusable initiators for synthesizing nucleic acids
US10683536B2 (en) 2013-04-02 2020-06-16 Molecular Assemblies, Inc. Reusable initiators for synthesizing nucleic acids
CN105849275B (zh) 2013-06-25 2020-03-17 普罗格诺西斯生物科学公司 检测样品中生物靶标的空间分布的方法和系统
US9352315B2 (en) 2013-09-27 2016-05-31 Taiwan Semiconductor Manufacturing Company, Ltd. Method to produce chemical pattern in micro-fluidic structure
US10540783B2 (en) * 2013-11-01 2020-01-21 Illumina, Inc. Image analysis useful for patterned objects
EP3077943B1 (fr) 2013-12-03 2020-06-03 Illumina, Inc. Procédés et systèmes d'analyse de données d'image
CN110411998B (zh) 2013-12-10 2022-06-07 伊鲁米那股份有限公司 用于生物或化学分析的生物传感器及其制造方法
WO2015103225A1 (fr) 2013-12-31 2015-07-09 Illumina, Inc. Cuve à circulation adressable utilisant des électrodes à motif
CN110955371B (zh) 2014-02-13 2023-09-12 Illumina公司 综合式消费者基因组服务
US10422002B2 (en) 2014-02-18 2019-09-24 Illumina, Inc. Methods and compositions for DNA profiling
CN106459869B (zh) 2014-03-11 2020-01-21 伊鲁米那股份有限公司 一次性的集成微流体盒及其制备和使用的方法
GB201408077D0 (en) 2014-05-07 2014-06-18 Illumina Cambridge Ltd Polymethine compounds and their use as fluorescent labels
SG10201809312QA (en) 2014-05-16 2018-11-29 Illumina Inc Nucleic acid synthesis techniques
RU2682546C2 (ru) 2014-05-27 2019-03-19 Иллумина, Инк. Системы и способы биохимического анализа, включающие основной прибор и съемный картридж
US20150353989A1 (en) 2014-06-09 2015-12-10 Illumina Cambridge Limited Sample preparation for nucleic acid amplification
PL3161154T3 (pl) * 2014-06-27 2020-10-19 Illumina, Inc. Zmodyfikowane polimerazy do ulepszonego włączania analogów nukleotydów
US9469862B2 (en) * 2014-08-19 2016-10-18 Arizona Board Of Regents On Behalf Of Arizona State University Modified polymerases for replication of threose nucleic acids
WO2016033315A2 (fr) 2014-08-27 2016-03-03 New England Biolabs, Inc. Formation de synthon
US9963687B2 (en) 2014-08-27 2018-05-08 New England Biolabs, Inc. Fusion polymerase and method for using the same
WO2016040602A1 (fr) 2014-09-11 2016-03-17 Epicentre Technologies Corporation Séquence au bisulfite à représentation réduite utilisant de l'uracile n-glycosylase (ung) et de l'endonucléase iv
WO2016054096A1 (fr) 2014-09-30 2016-04-07 Illumina, Inc. Polymérases modifiées pour l'incorporation améliorée d'analogues de nucléotides
US9897791B2 (en) 2014-10-16 2018-02-20 Illumina, Inc. Optical scanning systems for in situ genetic analysis
GB201419731D0 (en) 2014-11-05 2014-12-17 Illumina Cambridge Ltd Sequencing from multiple primers to increase data rate and density
ES2768762T3 (es) 2014-11-05 2020-06-23 Illumina Cambridge Ltd Reducción del daño al ADN durante la preparación de muestras y secuenciación usando agentes quelantes sideróforos
JP7032930B2 (ja) 2014-11-11 2022-03-09 イルミナ ケンブリッジ リミテッド 核酸のモノクローナルクラスターの生成および配列決定のための方法およびアレイ
WO2016077795A1 (fr) 2014-11-14 2016-05-19 Illumina, Inc. Polymérases
ES2786652T3 (es) 2015-02-10 2020-10-13 Illumina Inc Métodos y composiciones para analizar componentes celulares
WO2016154038A1 (fr) 2015-03-20 2016-09-29 Illumina, Inc. Cartouche fluidique pour une utilisation dans la position verticale ou sensiblement verticale
ES2846730T3 (es) 2015-03-31 2021-07-29 Illumina Cambridge Ltd Concatemerización en superficie de moldes
CA2982146A1 (fr) 2015-04-10 2016-10-13 Spatial Transcriptomics Ab Analyse de plusieurs acides nucleiques spatialement differencies de specimens biologiques
WO2016182984A1 (fr) 2015-05-08 2016-11-17 Centrillion Technology Holdings Corporation Terminateurs réversibles à liaison disulfure
GB201508858D0 (en) 2015-05-22 2015-07-01 Illumina Cambridge Ltd Polymethine compounds with long stokes shifts and their use as fluorescent labels
EP3303584B1 (fr) 2015-05-29 2019-10-09 Epicentre Technologies Corporation Méthodes d'analyse d'acides nucléiques
CA2985545C (fr) 2015-05-29 2021-02-09 Illumina Cambridge Limited Utilisation amelioree d'amorces de surface dans des amas
EP3320111B1 (fr) 2015-07-06 2021-05-05 Illumina Cambridge Limited Préparation d'échantillon pour l'amplification d'acide nucléique
DK3329012T3 (da) 2015-07-27 2021-10-11 Illumina Inc Rumlig kortlægning af nukleinsyresekvensinformation
PL3334839T3 (pl) 2015-08-14 2021-08-02 Illumina, Inc. Systemy i sposoby wykorzystujące czujniki reagujące na pole magnetyczne do określania informacji genetycznej
WO2017034868A1 (fr) 2015-08-24 2017-03-02 Illumina, Inc. Accumulateur de pression en ligne et système de commande d'écoulement pour dosages biologiques ou chimiques
US10450598B2 (en) 2015-09-11 2019-10-22 Illumina, Inc. Systems and methods for obtaining a droplet having a designated concentration of a substance-of-interest
GB201516987D0 (en) 2015-09-25 2015-11-11 Illumina Cambridge Ltd Polymethine compounds and their use as fluorescent labels
US10577643B2 (en) 2015-10-07 2020-03-03 Illumina, Inc. Off-target capture reduction in sequencing techniques
WO2017062965A1 (fr) * 2015-10-08 2017-04-13 Arizona Board Of Regents On Behalf Of Arizona State University Capteur optique hautement sensible destiné au criblage de polymérase
US10273539B2 (en) 2015-11-06 2019-04-30 Qiagen Sciences, Llc Methods of using nucleotide analogues
US11421264B2 (en) 2015-11-06 2022-08-23 IsoPlexis Corporation Thiol-containing cleave reagents and oxidative wash
US10253352B2 (en) 2015-11-17 2019-04-09 Omniome, Inc. Methods for determining sequence profiles
SG11201811504PA (en) 2016-07-22 2019-01-30 Univ Oregon Health & Science Single cell whole genome libraries and combinatorial indexing methods of making thereof
CA3213915A1 (fr) 2016-09-22 2018-03-29 Illumina, Inc. Detection de la variation du nombre de copies somatiques
US10385214B2 (en) 2016-09-30 2019-08-20 Illumina Cambridge Limited Fluorescent dyes and their uses as biomarkers
EP4439045A3 (fr) 2016-10-14 2024-11-27 Illumina, Inc. Ensemble cartouche
KR102512190B1 (ko) 2016-12-22 2023-03-21 일루미나 케임브리지 리미티드 쿠마린 화합물 및 형광 표지로서의 이의 용도
BR112019013715B1 (pt) 2017-01-04 2021-08-24 Bgi Shenzhen Métodos para detectar incorporação de um 3?-o-desoxirribonucleotídeo terminador reversível, para realizar uma reação de sequenciamento por síntese, para realizar sequenciamento por síntese de desoxirribonucleotídeo na extremidade 3? de um produto de extensão de iniciador e para sequenciamento de um ácido nucleico, arranjo de dna, e, kit
LT3566158T (lt) 2017-01-06 2022-06-27 Illumina, Inc. Fazinė korekcija
US20200090784A1 (en) 2017-01-17 2020-03-19 Illumina, Inc. Oncogenic splice variant determination
EP3571313B1 (fr) 2017-01-20 2020-12-23 Omniome, Inc. Capture spécifique d'un allèle d'acides nucléiques
CA3050852C (fr) 2017-01-20 2021-03-09 Omniome, Inc. Genotypage par liaison par polymerase
WO2018148724A1 (fr) 2017-02-13 2018-08-16 Qiagen Waltham Inc. Enzyme polymérase de pyrococcus furiosus
EP3580334A1 (fr) * 2017-02-13 2019-12-18 QIAGEN GmbH Enzyme polymérase issue du phage t4
EP3580350B1 (fr) 2017-02-13 2020-11-18 Qiagen Sciences, LLC Enzyme polymérase de pyrococcus furiosus
US20220145272A1 (en) * 2017-02-13 2022-05-12 IsoPlexis Corporation Polymerase enzyme from pyrococcus abyssi
WO2018148723A1 (fr) 2017-02-13 2018-08-16 Qiagen Waltham Inc. Enzyme polymérase de pyrococcus abyssi
WO2018148727A1 (fr) 2017-02-13 2018-08-16 Qiagen Waltham Inc. Enzyme polymérase de 9°n
US20200002689A1 (en) 2017-02-13 2020-01-02 Qiagen Sciences, Llc Polymerase enzyme from 9°n
WO2018148726A1 (fr) 2017-02-13 2018-08-16 Qiagen Waltham Inc. Enzyme polymérase issue du phage t4
US20200377935A1 (en) 2017-03-24 2020-12-03 Life Technologies Corporation Polynucleotide adapters and methods of use thereof
AU2018259206B2 (en) 2017-04-23 2024-07-11 Illumina Cambridge Limited Compositions and methods for improving sample identification in indexed nucleic acid libraries
SG11201909914RA (en) 2017-04-23 2019-11-28 Illumina Cambridge Ltd Compositions and methods for improving sample identification in indexed nucleic acid libraries
ES2937929T3 (es) 2017-04-23 2023-04-03 Illumina Inc Composiciones y métodos para mejorar la identificación de muestras en bibliotecas de ácido nucleico indexadas
SG10202108175RA (en) 2017-06-07 2021-09-29 Univ Oregon Health & Science Single cell whole genome libraries for methylation sequencing
WO2018236631A1 (fr) 2017-06-20 2018-12-27 Illumina, Inc. Procédés et compositions pour résoudre les inefficacités dans des réactions d'amplification
GB201711219D0 (en) 2017-07-12 2017-08-23 Illumina Cambridge Ltd Short pendant arm linkers for nucleotides in sequencing applications
EP3662482A1 (fr) 2017-07-31 2020-06-10 Illumina Inc. Système de séquençage comportant une agrégation d'échantillons biologiques multiplexés
CN111108219B (zh) 2017-08-01 2024-12-27 伊鲁米纳公司 使用的水凝胶珠和流动池的遗传物质的空间索引和文库制备
FI3663407T3 (fi) 2017-08-01 2023-04-18 Mgi Tech Co Ltd Nukleiinihapon sekvensointimenetelmä
JP6998404B2 (ja) 2017-08-01 2022-02-04 深▲セン▼恒特基因有限公司 標的ヌクレオチド配列の富化及び決定方法
EP3665274A1 (fr) 2017-08-07 2020-06-17 DNA Script Variants de l'adn polymérase de la famille a et leurs utilisations
US20190078126A1 (en) * 2017-09-08 2019-03-14 Sigma-Aldrich Co. Llc Polymerase-mediated, template-independent polynucleotide synthesis
UA128674C2 (uk) 2017-09-20 2024-09-25 Рідженерон Фармасьютикалз, Інк. Імунотерапія пацієнта, пухлина у якого характеризується високим навантаженням пасажирськими генними мутаціями
GB201716931D0 (en) 2017-10-16 2017-11-29 Illumina Cambridge Ltd New fluorescent compounds and their use as biomarkers
CA3202587A1 (fr) 2017-11-06 2019-05-09 Illumina, Inc Techniques d'indexation d'acide nucleique
KR102402002B1 (ko) 2017-11-16 2022-05-25 일루미나, 인코포레이티드 미세부수체 불안정성을 결정하기 위한 시스템 및 방법
JP7013490B2 (ja) 2017-11-30 2022-02-15 イルミナ インコーポレイテッド 配列バリアントコールのためのバリデーションの方法及びシステム
US11561196B2 (en) 2018-01-08 2023-01-24 Illumina, Inc. Systems and devices for high-throughput sequencing with semiconductor-based detection
US10752887B2 (en) 2018-01-08 2020-08-25 Dna Script Variants of terminal deoxynucleotidyl transferase and uses thereof
KR102239487B1 (ko) 2018-01-08 2021-04-14 일루미나, 인코포레이티드 반도체-기반 검출을 사용한 고-처리율 서열분석
US10844430B2 (en) 2018-01-24 2020-11-24 Qiagen Sciences, Llc DNA sequencing reaction additive
MX2019014803A (es) 2018-03-09 2021-02-09 Illumina Cambridge Ltd Secuenciaciòn estocástica generalizada de super-resolución.
US12188012B2 (en) 2018-04-02 2025-01-07 Illumina, Inc. Compositions and methods for making controls for sequence-based genetic testing
WO2019222688A1 (fr) 2018-05-17 2019-11-21 Illumina, Inc. Séquençage de cellules uniques à haut débit avec biais d'amplification réduit
KR102507415B1 (ko) 2018-06-04 2023-03-07 일루미나, 인코포레이티드 고속 대량 단일 세포 전사체 라이브러리 그리고 이의 제조 및 사용 방법
NL2021377B1 (en) 2018-07-03 2020-01-08 Illumina Inc Interposer with first and second adhesive layers
US20210317524A1 (en) 2018-08-28 2021-10-14 10X Genomics, Inc. Resolving spatial arrays
US11519033B2 (en) 2018-08-28 2022-12-06 10X Genomics, Inc. Method for transposase-mediated spatial tagging and analyzing genomic DNA in a biological sample
CN113767175A (zh) 2018-08-28 2021-12-07 10X基因组学股份有限公司 增加空间阵列分辨率
US20230011240A1 (en) 2018-09-11 2023-01-12 Singular Genomics Systems, Inc. Modified archaeal family b polymerases
US10731141B2 (en) 2018-09-17 2020-08-04 Omniome, Inc. Engineered polymerases for improved sequencing
EP3856753A4 (fr) 2018-09-28 2022-11-16 Centrillion Technology Holdings Corporation Terminateurs réversibles à liaison disulfure
CA3103719A1 (fr) * 2018-10-31 2020-05-07 Illumina, Inc. Polymerases, compositions et procedes d'utilisation
NL2022043B1 (en) 2018-11-21 2020-06-03 Akershus Univ Hf Tagmentation-Associated Multiplex PCR Enrichment Sequencing
US20220025447A1 (en) 2018-12-10 2022-01-27 10X Genomics, Inc. Generating spatial arrays with gradients
US11293061B2 (en) 2018-12-26 2022-04-05 Illumina Cambridge Limited Sequencing methods using nucleotides with 3′ AOM blocking group
US11926867B2 (en) 2019-01-06 2024-03-12 10X Genomics, Inc. Generating capture probes for spatial analysis
US11649485B2 (en) 2019-01-06 2023-05-16 10X Genomics, Inc. Generating capture probes for spatial analysis
CN114174531A (zh) 2019-02-28 2022-03-11 10X基因组学有限公司 用空间条码化寡核苷酸阵列对生物分析物进行概况分析
CN112654614A (zh) 2019-03-01 2021-04-13 伊卢米纳剑桥有限公司 叔胺取代的香豆素化合物及其作为荧光标记物的用途
US20220033805A1 (en) 2019-03-01 2022-02-03 Sanjay Srivatsan High-throughput single-nuclei and single-cell libraries and methods of making and of using
WO2020178231A1 (fr) 2019-03-01 2020-09-10 Illumina, Inc. Détection fluorescente multiplexée d'analytes
KR20210134209A (ko) 2019-03-01 2021-11-09 일루미나 케임브리지 리미티드 엑소사이클릭 아민 치환된 쿠마린 화합물 및 형광 표지로서의 이의 용도
NL2023327B1 (en) 2019-03-01 2020-09-17 Illumina Inc Multiplexed fluorescent detection of analytes
CN114127309A (zh) 2019-03-15 2022-03-01 10X基因组学有限公司 使用空间阵列进行单细胞测序的方法
NL2023310B1 (en) 2019-03-21 2020-09-28 Illumina Inc Training data generation for artificial intelligence-based sequencing
NL2023314B1 (en) 2019-03-21 2020-09-28 Illumina Inc Artificial intelligence-based quality scoring
WO2020191387A1 (fr) 2019-03-21 2020-09-24 Illumina, Inc. Appel de base à base d'intelligence artificielle
NL2023311B9 (en) 2019-03-21 2021-03-12 Illumina Inc Artificial intelligence-based generation of sequencing metadata
NL2023312B1 (en) 2019-03-21 2020-09-28 Illumina Inc Artificial intelligence-based base calling
US11210554B2 (en) 2019-03-21 2021-12-28 Illumina, Inc. Artificial intelligence-based generation of sequencing metadata
US11783917B2 (en) 2019-03-21 2023-10-10 Illumina, Inc. Artificial intelligence-based base calling
NL2023316B1 (en) 2019-03-21 2020-09-28 Illumina Inc Artificial intelligence-based sequencing
EP3887542A1 (fr) 2019-03-22 2021-10-06 10X Genomics, Inc. Analyse spatiale tridimensionnelle
US11421271B2 (en) 2019-03-28 2022-08-23 Illumina Cambridge Limited Methods and compositions for nucleic acid sequencing using photoswitchable labels
US11423306B2 (en) 2019-05-16 2022-08-23 Illumina, Inc. Systems and devices for characterization and performance analysis of pixel-based sequencing
US11593649B2 (en) 2019-05-16 2023-02-28 Illumina, Inc. Base calling using convolutions
WO2020243579A1 (fr) 2019-05-30 2020-12-03 10X Genomics, Inc. Procédés de détection de l'hétérogénéité spatiale d'un échantillon biologique
US11512295B2 (en) 2019-09-12 2022-11-29 Singular Genomics Systems, Inc. Modified thermoccocus polymerases
CA3151411A1 (fr) 2019-09-23 2021-04-01 Adrian Horgan Augmentation de rendements de sequence longue dans la synthese enzymatique sans matrice de polynucleotides
CN117036248A (zh) 2019-10-01 2023-11-10 10X基因组学有限公司 用于识别组织样品中的形态学模式的系统和方法
WO2021092433A2 (fr) 2019-11-08 2021-05-14 10X Genomics, Inc. Amélioration de la spécificité de la liaison d'un analyte
EP4055185A1 (fr) 2019-11-08 2022-09-14 10X Genomics, Inc. Agents de capture d'analytes marqués spatialement pour le multiplexage d'analytes
US20210139867A1 (en) 2019-11-08 2021-05-13 Omniome, Inc. Engineered polymerases for improved sequencing by binding
CN115004260A (zh) 2019-11-18 2022-09-02 10X基因组学有限公司 用于组织分类的系统和方法
CN117078725A (zh) 2019-11-21 2023-11-17 10X基因组学有限公司 分析物的空间分析
JP2023510454A (ja) 2019-11-27 2023-03-14 イルミナ ケンブリッジ リミテッド シクロオクタテトラエンを含有する色素及び組成物
CN114829368B (zh) 2019-12-18 2024-06-04 豪夫迈·罗氏有限公司 通过使用连续标记方案的合成进行测序的方法
CN114008199A (zh) 2019-12-19 2022-02-01 伊路敏纳公司 高通量单细胞文库及其制备和使用方法
EP4081656A1 (fr) 2019-12-23 2022-11-02 10X Genomics, Inc. Compositions et méthodes d'utilisation d'échantillons biologiques fixés dans des dosages basés sur des compartiments
EP4424843A3 (fr) 2019-12-23 2024-09-25 10X Genomics, Inc. Procédés d'analyse spatiale à l'aide d'une ligature à matrice d'arn
CN110938611B (zh) * 2019-12-31 2021-05-04 莫纳(武汉)生物科技有限公司 一种dna聚合酶及其制备方法和应用
US11732299B2 (en) 2020-01-21 2023-08-22 10X Genomics, Inc. Spatial assays with perturbed cells
US11702693B2 (en) 2020-01-21 2023-07-18 10X Genomics, Inc. Methods for printing cells and generating arrays of barcoded cells
US12076701B2 (en) 2020-01-31 2024-09-03 10X Genomics, Inc. Capturing oligonucleotides in spatial transcriptomics
US11898205B2 (en) 2020-02-03 2024-02-13 10X Genomics, Inc. Increasing capture efficiency of spatial assays
US12110541B2 (en) 2020-02-03 2024-10-08 10X Genomics, Inc. Methods for preparing high-resolution spatial arrays
US12112833B2 (en) 2020-02-04 2024-10-08 10X Genomics, Inc. Systems and methods for index hopping filtering
US11732300B2 (en) 2020-02-05 2023-08-22 10X Genomics, Inc. Increasing efficiency of spatial analysis in a biological sample
US12129516B2 (en) 2020-02-07 2024-10-29 10X Genomics, Inc. Quantitative and automated permeabilization performance evaluation for spatial transcriptomics
US12215379B2 (en) 2020-02-17 2025-02-04 10X Genomics, Inc. In situ analysis of chromatin interaction
US20210265015A1 (en) 2020-02-20 2021-08-26 Illumina, Inc. Hardware Execution and Acceleration of Artificial Intelligence-Based Base Caller
IL295560A (en) 2020-02-20 2022-10-01 Illumina Inc Artificial intelligence-based many-to-many base calling
US20210265016A1 (en) 2020-02-20 2021-08-26 Illumina, Inc. Data Compression for Artificial Intelligence-Based Base Calling
US20210265018A1 (en) 2020-02-20 2021-08-26 Illumina, Inc. Knowledge Distillation and Gradient Pruning-Based Compression of Artificial Intelligence-Based Base Caller
EP4484571A3 (fr) 2020-02-21 2025-03-19 10X Genomics, Inc. Procédés et compositions pour un dosage spatial in situ intégré
US11891654B2 (en) 2020-02-24 2024-02-06 10X Genomics, Inc. Methods of making gene expression libraries
US11034942B1 (en) 2020-02-27 2021-06-15 Singular Genomics Systems, Inc. Modified pyrococcus polymerases and uses thereof
US12188085B2 (en) 2020-03-05 2025-01-07 10X Genomics, Inc. Three-dimensional spatial transcriptomics with sequencing readout
EP4121554B1 (fr) 2020-03-18 2024-10-30 MGI Tech Co., Ltd. Restauration de phase dans un séquençage massivement parallèle
EP4139485B1 (fr) 2020-04-22 2023-09-06 10X Genomics, Inc. Procédés d'analyse spatiale utilisant un appauvrissement d'arn ciblée
US20230203592A1 (en) 2020-05-05 2023-06-29 Akershus Universitetssykehus Hf Compositions and methods for characterizing bowel cancer
US11188778B1 (en) 2020-05-05 2021-11-30 Illumina, Inc. Equalization-based image processing and spatial crosstalk attenuator
JP2023524531A (ja) 2020-05-08 2023-06-12 イルミナ インコーポレイテッド ゲノムシーケンシング及び検出手法
CN115916967A (zh) 2020-05-12 2023-04-04 伊鲁米纳公司 使用重组末端脱氧核苷酸转移酶生成具有经修饰的碱基的核酸
ES2989052T3 (es) 2020-05-22 2024-11-25 10X Genomics Inc Medición espacio-temporal simultánea de la expresión génica y la actividad celular
EP4153776B1 (fr) 2020-05-22 2025-03-05 10X Genomics, Inc. Analyse spatiale pour détecter des variants de séquence
WO2021242834A1 (fr) 2020-05-26 2021-12-02 10X Genomics, Inc. Procédé de réinitialisation d'un réseau
US12031177B1 (en) 2020-06-04 2024-07-09 10X Genomics, Inc. Methods of enhancing spatial resolution of transcripts
EP4421186A3 (fr) 2020-06-08 2024-09-18 10X Genomics, Inc. Procédés de détermination d'une marge chirurgicale et procédés d'utilisation de ceux-ci
WO2021252617A1 (fr) 2020-06-09 2021-12-16 Illumina, Inc. Procédés pour augmenter le rendement de bibliothèques de séquençage
EP4165207B1 (fr) 2020-06-10 2024-09-25 10X Genomics, Inc. Methode pour determiner la position d'un analyte dans un echantillon biologique
MA58991B1 (fr) 2020-06-22 2024-09-30 Illumina Cambridge Limited Nucléosides et nucléotides avec groupe de blocage acétal 3'
AU2021294334A1 (en) 2020-06-25 2023-02-02 10X Genomics, Inc. Spatial analysis of DNA methylation
CN115997033A (zh) 2020-06-30 2023-04-21 伊鲁米纳公司 通过合成产生无痕dna的催化控制测序
US12168801B1 (en) 2020-07-02 2024-12-17 10X Genomics, Inc. Hybrid/capture probe designs for full-length cDNA
US12209280B1 (en) 2020-07-06 2025-01-28 10X Genomics, Inc. Methods of identifying abundance and location of an analyte in a biological sample using second strand synthesis
US11761038B1 (en) 2020-07-06 2023-09-19 10X Genomics, Inc. Methods for identifying a location of an RNA in a biological sample
US11981960B1 (en) 2020-07-06 2024-05-14 10X Genomics, Inc. Spatial analysis utilizing degradable hydrogels
KR20230038145A (ko) 2020-07-08 2023-03-17 일루미나, 인코포레이티드 트랜스포좀 담체로서의 비드
US11981964B2 (en) 2020-07-28 2024-05-14 Illumina Cambridge Limited Substituted coumarin dyes and uses as fluorescent labels
US11981958B1 (en) 2020-08-20 2024-05-14 10X Genomics, Inc. Methods for spatial analysis using DNA capture
US20220067489A1 (en) 2020-08-28 2022-03-03 Illumina, Inc. Detecting and Filtering Clusters Based on Artificial Intelligence-Predicted Base Calls
US11926822B1 (en) 2020-09-23 2024-03-12 10X Genomics, Inc. Three-dimensional spatial analysis
US12071667B2 (en) 2020-11-04 2024-08-27 10X Genomics, Inc. Sequence analysis using meta-stable nucleic acid molecules
US11827935B1 (en) 2020-11-19 2023-11-28 10X Genomics, Inc. Methods for spatial analysis using rolling circle amplification and detection probes
WO2022119812A1 (fr) 2020-12-02 2022-06-09 Illumina Software, Inc. Système et procédé pour la détection d'altérations génétiques
US20220186300A1 (en) 2020-12-11 2022-06-16 10X Genomics, Inc. Methods and compositions for multimodal in situ analysis
US20220195517A1 (en) 2020-12-17 2022-06-23 Illumina Cambridge Limited Long stokes shift chromenoquinoline dyes and uses in sequencing applications
US20220195196A1 (en) 2020-12-17 2022-06-23 Illumina Cambridge Limited Alkylpyridinium coumarin dyes and uses in sequencing applications
US20220195516A1 (en) 2020-12-17 2022-06-23 Illumina Cambridge Limited Methods, systems and compositions for nucleic acid sequencing
WO2022140028A1 (fr) 2020-12-21 2022-06-30 10X Genomics, Inc. Procédés, compositions et systèmes pour capturer des sondes et/ou des codes à barres
US20220195518A1 (en) 2020-12-22 2022-06-23 Illumina Cambridge Limited Methods and compositions for nucleic acid sequencing
US12060603B2 (en) 2021-01-19 2024-08-13 10X Genomics, Inc. Methods for internally controlled in situ assays using padlock probes
EP4284945B1 (fr) 2021-01-26 2024-10-23 10X Genomics, Inc. Sondes analogiques d'acide nucléique pour analyse in situ
WO2022187366A1 (fr) 2021-03-03 2022-09-09 10X Genomics, Inc. Détection d'analytes in situ à l'aide d'origami d'acide nucléique
WO2022197754A1 (fr) 2021-03-16 2022-09-22 Illumina Software, Inc. Quantification de paramètres de réseau neuronal pour appel de base
US20240287481A1 (en) * 2021-03-19 2024-08-29 Mgi Tech Co., Ltd. Polymerases for efficient incorporation of nucleotides with 3'-phosphate and other 3'-terminators
BR112023019154A2 (pt) 2021-03-31 2023-10-17 Illumina Cambridge Ltd Técnicas de sequenciamento de bibliotecas de ácidos nucleicos com detecção de dímero de adaptador
AU2022248999A1 (en) 2021-03-31 2023-02-02 Illumina, Inc. Artificial intelligence-based base caller with contextual awareness
US20220319641A1 (en) 2021-04-02 2022-10-06 Illumina, Inc. Machine-learning model for detecting a bubble within a nucleotide-sample slide for sequencing
EP4305196B1 (fr) 2021-04-14 2025-04-02 10X Genomics, Inc. Procédés de mesure d'une mauvaise localisation d'un analyte
US20220333146A1 (en) 2021-04-14 2022-10-20 Illumina Singapore Pte. Ltd. Synthesis of polynucleotide bottlebrush polymer
US12217829B2 (en) 2021-04-15 2025-02-04 Illumina, Inc. Artificial intelligence-based analysis of protein three-dimensional (3D) structures
FR3121936A1 (fr) 2021-04-20 2022-10-21 Naos Institute Of Life Science Methode de criblage d’ingredients et de compositions topiques ecobiologiques, et son utilisation pour realiser un soin cosmetique de restauration du microbiote cutane
US20240218436A1 (en) 2021-04-29 2024-07-04 Illumina, Inc. Amplification techniques for nucleic acid characterization
WO2022233795A1 (fr) 2021-05-05 2022-11-10 Illumina Cambridge Limited Colorants fluorescents contenant des hétérocycles fusionnés contenant du bis-bore et leurs utilisations dans le séquençage
BR112023024130A2 (pt) 2021-05-20 2024-01-30 Illumina Cambridge Ltd Composições e métodos para sequenciamento por síntese
US20220380838A1 (en) 2021-06-01 2022-12-01 10X Genomics, Inc. Methods and compositions for analyte detection and probe resolution
EP4347880A1 (fr) 2021-06-02 2024-04-10 10X Genomics, Inc. Analyse d'échantillon à l'aide de sondes circularisables asymétriques
EP4347879B1 (fr) 2021-06-03 2025-02-19 10X Genomics, Inc. Procédés, compositions, kits et systèmes pour améliorer la capture d'analytes pour une analyse spatiale
US20220403450A1 (en) 2021-06-03 2022-12-22 Illumina Software, Inc. Systems and methods for sequencing nucleotides using two optical channels
US12270056B2 (en) 2021-06-18 2025-04-08 Element Biosciences, Inc. Engineered polymerases
EP4355867A2 (fr) * 2021-06-18 2024-04-24 Element Biosciences, Inc. Polymérases modifiées
EP4359555A1 (fr) 2021-06-23 2024-05-01 Illumina, Inc. Compositions, procédés, kits, cartouches et systèmes de séquençage de réactifs
CA3224393A1 (fr) 2021-06-29 2023-01-05 Mitchell A BEKRITSKY Modele d'apprentissage automatique pour generer des classifications de confiance pour des coordonnees genomiques
EP4364150A1 (fr) 2021-06-29 2024-05-08 Illumina, Inc. Appelant de base auto-appris, entraîné à l'aide de séquences d'organismes
JP2024527307A (ja) 2021-06-29 2024-07-24 イルミナ インコーポレイテッド ヌクレオチド塩基コール及び塩基コール品質を決定するための信号対雑音比メトリック
WO2023278184A1 (fr) 2021-06-29 2023-01-05 Illumina, Inc. Procédés et systèmes pour corriger une diaphonie dans un éclairage émis par des sites de réaction
US20230005253A1 (en) 2021-07-01 2023-01-05 Illumina, Inc. Efficient artificial intelligence-based base calling of index sequences
EP4370896A1 (fr) 2021-07-13 2024-05-22 10X Genomics, Inc. Procédés de préparation d'une matrice polymérisée à épaisseur contrôlable
US20230027409A1 (en) 2021-07-13 2023-01-26 Illumina, Inc. Methods and systems for real time extraction of crosstalk in illumination emitted from reaction sites
JP2024532004A (ja) 2021-07-19 2024-09-04 イルミナ インコーポレイテッド ベースコールのための補間及び適合による強度抽出
US11455487B1 (en) 2021-10-26 2022-09-27 Illumina Software, Inc. Intensity extraction and crosstalk attenuation using interpolation and adaptation for base calling
US20230021577A1 (en) 2021-07-23 2023-01-26 Illumina Software, Inc. Machine-learning model for recalibrating nucleotide-base calls
AU2022319125A1 (en) 2021-07-28 2024-01-18 Illumina, Inc. Quality score calibration of basecalling systems
US20230041485A1 (en) 2021-07-30 2023-02-09 10X Genomics, Inc. Methods and compositions for synchronizing reactions in situ
US12139751B2 (en) 2021-07-30 2024-11-12 10X Genomics, Inc. Circularizable probes for in situ analysis
JP2024529843A (ja) 2021-08-03 2024-08-14 イルミナ インコーポレイテッド 複数のベースコーラモデルを使用するベースコール
EP4381095A1 (fr) 2021-08-03 2024-06-12 10X Genomics, Inc. Concatémères d'acide nucléique et procédés de stabilisation et/ou de compactage de ceux-ci
CN117858958A (zh) 2021-08-16 2024-04-09 10X基因组学有限公司 包含分割条形码区的探针和使用方法
WO2023034489A1 (fr) 2021-09-01 2023-03-09 10X Genomics, Inc. Procédés, compositions et kits pour bloquer une sonde de capture sur un réseau spatial
CN117580961A (zh) 2021-09-01 2024-02-20 Illumina公司 用于加速碱基判读的幅度调制
US20230093253A1 (en) 2021-09-17 2023-03-23 Illumina, Inc. Automatically identifying failure sources in nucleotide sequencing from base-call-error patterns
US20230095961A1 (en) 2021-09-21 2023-03-30 Illumina, Inc. Graph reference genome and base-calling approach using imputed haplotypes
WO2023049212A2 (fr) 2021-09-22 2023-03-30 Illumina, Inc. Appel de base basé sur l'état
WO2023102118A2 (fr) 2021-12-01 2023-06-08 10X Genomics, Inc. Procédés, compositions et systèmes pour la détection améliorée d'analytes in situ et analyse spatiale
CN117581303A (zh) 2021-12-02 2024-02-20 因美纳有限公司 产生用于确定核苷酸碱基检出的簇特异性信号校正
WO2023107622A1 (fr) 2021-12-10 2023-06-15 Illumina, Inc. Séquençage d'échantillons et d'indices parallèles
WO2023114896A1 (fr) 2021-12-16 2023-06-22 Illumina Cambridge Limited Procédés de clivage dirigé par métal de polynucléotides liés à une surface
US20240401127A1 (en) 2021-12-20 2024-12-05 Illumina, Inc. Periodate compositions and methods for chemical cleavage of surface-bound polynucleotides
CN117813399A (zh) 2021-12-20 2024-04-02 因美纳有限公司 用于化学裂解表面结合的多核苷酸的高碘酸盐组合物和方法
US20230215515A1 (en) 2021-12-23 2023-07-06 Illumina Software, Inc. Facilitating secure execution of external workflows for genomic sequencing diagnostics
WO2023122363A1 (fr) 2021-12-23 2023-06-29 Illumina Software, Inc. Résumés dynamiques d'état graphique pour séquençage de nucléotides
EP4423296A2 (fr) 2021-12-27 2024-09-04 10X Genomics, Inc. Procédés et compositions pour l'amplification par cercle roulant
US20230207050A1 (en) 2021-12-28 2023-06-29 Illumina Software, Inc. Machine learning model for recalibrating nucleotide base calls corresponding to target variants
WO2023129764A1 (fr) 2021-12-29 2023-07-06 Illumina Software, Inc. Permutation automatique des versions de modèle d'analyse de variants pour les applications d'analyse génomique
WO2023141588A1 (fr) 2022-01-21 2023-07-27 10X Genomics, Inc. Signaux de lecture multiples pour analyser un échantillon
US11788075B2 (en) 2022-02-18 2023-10-17 Element Biosciences, Inc. Engineered polymerases with reduced sequence-specific errors
AU2023223467A1 (en) 2022-02-23 2024-08-08 Insitro, Inc. Pooled optical screening and transcriptional measurements of cells comprising barcoded genetic perturbations
AU2023225949A1 (en) 2022-02-25 2024-01-18 Illumina, Inc. Machine-learning models for detecting and adjusting values for nucleotide methylation levels
EP4483372A1 (fr) 2022-02-25 2025-01-01 Illumina, Inc. Séquences d'étalonnage à des fins de séquençage de nucléotide
KR20240162122A (ko) 2022-03-15 2024-11-14 일루미나, 인코포레이티드 병렬 샘플 및 인덱스 시퀀싱
WO2023183937A1 (fr) 2022-03-25 2023-09-28 Illumina, Inc. Appel de bases séquence par séquence
AU2023246676A1 (en) 2022-03-28 2024-01-18 Illumina Inc. Labeled avidin and methods for sequencing
WO2023186819A1 (fr) 2022-03-29 2023-10-05 Illumina Cambridge Limited Colorants de chroménoquinoléine et leurs utilisations dans le séquencage
US20230313294A1 (en) 2022-03-30 2023-10-05 Illumina Cambridge Limited Methods for chemical cleavage of surface-bound polynucleotides
EP4499870A1 (fr) 2022-03-31 2025-02-05 Illumina, Inc. Compositions et procédés pour améliorer des signaux de séquençage
US20230332197A1 (en) 2022-03-31 2023-10-19 Illumina Singapore Pte. Ltd. Nucleosides and nucleotides with 3' vinyl blocking group
WO2023192616A1 (fr) 2022-04-01 2023-10-05 10X Genomics, Inc. Compositions et procédés de masquage ciblé d'autofluorescence
EP4504969A1 (fr) 2022-04-06 2025-02-12 10X Genomics, Inc. Procédés d'analyse multiplex de cellules
KR20250004634A (ko) 2022-04-07 2025-01-08 일루미나, 인코포레이티드 변경된 시티딘 데아미나제 및 사용 방법
AU2023250540A1 (en) 2022-04-08 2024-01-18 Illumina, Inc. Aptamer dynamic range compression and detection techniques
US20230340571A1 (en) 2022-04-26 2023-10-26 Illumina, Inc. Machine-learning models for selecting oligonucleotide probes for array technologies
WO2023209606A1 (fr) 2022-04-29 2023-11-02 Illumina Cambridge Limited Procédés et systèmes d'encapsulation de microsphères lyophilisées
EP4519674A1 (fr) 2022-05-06 2025-03-12 10X Genomics, Inc. Analyse d'un antigène et des interactions antigène-récepteur
US20230368866A1 (en) 2022-05-10 2023-11-16 Illumina Software, Inc. Adaptive neural network for nucelotide sequencing
WO2023225095A1 (fr) 2022-05-18 2023-11-23 Illumina Cambridge Limited Préparation de fragments d'acide nucléique à taille contrôlée
US20230383342A1 (en) 2022-05-31 2023-11-30 Illumina Cambridge Limited Compositions and methods for nucleic acid sequencing
US20240035071A1 (en) 2022-06-17 2024-02-01 10X Genomics, Inc. Catalytic de-crosslinking of samples for in situ analysis
KR20250034034A (ko) 2022-06-24 2025-03-10 일루미나, 인코포레이티드 후보 분할 그룹을 지능적으로 식별하고 채점함으로써 분할-판독 정렬을 개선하는 방법
WO2024006705A1 (fr) 2022-06-27 2024-01-04 Illumina Software, Inc. Génotypage amélioré d'antigène leucocytaire humain (hla)
KR20250028287A (ko) 2022-06-27 2025-02-28 일루미나, 인코포레이티드 구조 변이 그래프 게놈의 생성 및 구현
US20230420075A1 (en) 2022-06-27 2023-12-28 Illumina Software, Inc. Accelerators for a genotype imputation model
CN119604513A (zh) 2022-06-28 2025-03-11 因美纳公司 含有稠合四环双硼杂环的荧光染料及其在测序中的用途
US20240038327A1 (en) 2022-07-26 2024-02-01 Illumina Software, Inc. Rapid single-cell multiomics processing using an executable file
CN119677856A (zh) 2022-08-12 2025-03-21 10X基因组学有限公司 Puma1聚合酶及其用途
US12116626B2 (en) 2022-08-16 2024-10-15 10X Genomics, Inc. AP50 polymerases and uses thereof
WO2024039516A1 (fr) 2022-08-19 2024-02-22 Illumina, Inc. Détection de la troisième paire de bases de l'adn spécifique de site
AU2023342375A1 (en) 2022-09-16 2025-01-16 Illumina, Inc. Cluster segmentation and conditional base calling
US20240112753A1 (en) 2022-09-29 2024-04-04 Illumina, Inc. Target-variant-reference panel for imputing target variants
WO2024073047A1 (fr) 2022-09-30 2024-04-04 Illumina, Inc. Cytidine désaminases et procédés d'utilisation dans la cartographie de nucléotides cytosine modifiés
US20240141427A1 (en) 2022-09-30 2024-05-02 Illumina, Inc. Polymerases, compositions, and methods of use
WO2024073043A1 (fr) 2022-09-30 2024-04-04 Illumina, Inc. Procédés d'utilisation de protéines de liaison cpg dans la cartographie de nucléotides cytosine modifiés
WO2024069581A1 (fr) 2022-09-30 2024-04-04 Illumina Singapore Pte. Ltd. Complexes hélicase-cytidine désaminase et procédés d'utilisation
WO2024068889A2 (fr) 2022-09-30 2024-04-04 Illumina, Inc. Compositions et procédés de réduction de photolésions pendant le séquençage
WO2024073519A1 (fr) 2022-09-30 2024-04-04 Illumina, Inc. Modèle d'apprentissage automatique pour affiner des appels de variants structuraux
CN119096301A (zh) 2022-10-05 2024-12-06 因美纳有限公司 利用机器学习架构集成来自多个测序管道的变体检出
US20240127906A1 (en) 2022-10-11 2024-04-18 Illumina, Inc. Detecting and correcting methylation values from methylation sequencing assays
US20240191297A1 (en) 2022-10-14 2024-06-13 10X Genomics, Inc. Methods, compositions, and systems for assessing biological sample quality
US20240167081A1 (en) 2022-11-08 2024-05-23 10X Genomics,Inc. Immobilization methods and compositions for in situ detection
WO2024107887A1 (fr) 2022-11-16 2024-05-23 10X Genomics, Inc. Procédés et compositions pour évaluer les performances de dosages in situ
US20240177802A1 (en) 2022-11-30 2024-05-30 Illumina, Inc. Accurately predicting variants from methylation sequencing data
US20240240217A1 (en) 2022-12-09 2024-07-18 Illumina, Inc. Nucleosides and nucleotides with 3' blocking groups and cleavable linkers
WO2024130203A1 (fr) 2022-12-16 2024-06-20 10X Genomics, Inc. Procédés et compositions d'évaluation de performances
WO2024137860A1 (fr) 2022-12-21 2024-06-27 10X Genomics, Inc. Procédés d'accroche d'acides ribonucléiques dans des échantillons biologiques
US20240212791A1 (en) 2022-12-21 2024-06-27 Illumina, Inc. Context-dependent base calling
CN119095982A (zh) 2022-12-22 2024-12-06 伊路米纳有限公司 用于边合成边测序的钯催化剂组合物和方法
WO2024137765A1 (fr) 2022-12-22 2024-06-27 Illumina, Inc. Compositions de catalyseur de métal de transition et procédés de séquençage par synthèse
CN119095984A (zh) 2022-12-27 2024-12-06 伊路米纳有限公司 使用3'烯丙基封端的核苷酸的测序方法
US20240263219A1 (en) 2023-01-06 2024-08-08 10X Genomics, Inc. Methods and compositions for in situ analysis of variant sequences
CN119452420A (zh) 2023-02-06 2025-02-14 因美纳有限公司 确定和去除簇间光干扰
AU2024235961A1 (en) 2023-03-10 2025-01-09 Illumina, Inc. Aptamer detection techniques
US20240327910A1 (en) 2023-03-29 2024-10-03 Illumina, Inc. Naphthalimide dyes and uses in nucleic acid sequencing
US20240327909A1 (en) 2023-03-30 2024-10-03 Illumina, Inc. Compositions and methods for nucleic acid sequencing
WO2024206848A1 (fr) 2023-03-30 2024-10-03 Illumina, Inc. Génotypage à répétition en tandem
US20240368678A1 (en) 2023-05-03 2024-11-07 10X Genomics, Inc. Methods and compositions for spatial assay
WO2024229396A1 (fr) 2023-05-03 2024-11-07 Illumina, Inc. Modèle d'apprentissage automatique pour réétalonner des appels de génotype à partir de fichiers de données de séquençage existants
WO2024249200A1 (fr) 2023-05-26 2024-12-05 Illumina, Inc. Procédés de conservation de l'état de méthylation pendant le regroupement
WO2024249940A1 (fr) 2023-05-31 2024-12-05 Illumina, Inc. Amélioration d'un alignement de variants structuraux et appel de variant à l'aide d'un génome de référence de variant structural
WO2024249973A2 (fr) 2023-06-02 2024-12-05 Illumina, Inc. Liaison de gènes humains à des phénotypes cliniques à l'aide de réseaux neuronaux graphiques
WO2025006565A1 (fr) 2023-06-27 2025-01-02 Illumina, Inc. Appel de variant avec estimation du niveau de méthylation
WO2025006464A1 (fr) 2023-06-30 2025-01-02 Illumina, Inc. Systèmes et procédés de séquençage de polynucléotides avec des diagrammes de dispersion alternatifs
WO2025006466A1 (fr) 2023-06-30 2025-01-02 Illumina, Inc. Systèmes et procédés de séquençage de polynucléotides avec quatre nucléotides marqués
WO2025006874A1 (fr) 2023-06-30 2025-01-02 Illumina, Inc. Modèle d'apprentissage automatique pour réétalonner des appels de génotype correspondant à des variants de lignée germinale et variants de mosaïque somatique
WO2025006460A1 (fr) 2023-06-30 2025-01-02 Illumina, Inc. Systèmes et procédés de séquençage de polynucléotides à bases modifiées
WO2025029831A1 (fr) 2023-07-31 2025-02-06 10X Genomics, Inc. Procédés et systèmes de clivage d'arn ciblé et d'amplification de cercle roulant amorcé par arn cible
WO2025049331A2 (fr) 2023-08-31 2025-03-06 Illumina, Inc. Techniques de détection d'aptamères
WO2025049700A1 (fr) 2023-08-31 2025-03-06 Illumina, Inc. Compositions et procédés de séquençage d'acides nucléiques
WO2025049720A2 (fr) 2023-08-31 2025-03-06 Illumina, Inc. Techniques de détection et de compression de plage dynamique d'aptamères
WO2025054389A1 (fr) 2023-09-07 2025-03-13 Illumina, Inc. Identification de la cytosine méthylée par l'utilisation de points de repère
EP4520821A1 (fr) 2023-09-08 2025-03-12 The Regents Of The University Of Michigan Arn et polypeptides dérivés de microarn et leurs utilisations
WO2025061942A1 (fr) 2023-09-20 2025-03-27 Illumina, Inc. Identification et correction d'erreur de séquençage
WO2025061922A1 (fr) 2023-09-20 2025-03-27 Illumina, Inc. Procédés de séquençage
WO2025072800A2 (fr) 2023-09-28 2025-04-03 Illumina, Inc. Cytidine désaminases modifiées et procédés d'utilisation
WO2025072870A1 (fr) 2023-09-29 2025-04-03 Illumina, Inc. Suivi et modification d'emplacement de cluster sur des lames d'échantillon de nucléotides en temps réel
WO2025072833A1 (fr) 2023-09-29 2025-04-03 Illumina, Inc. Prédiction de longueurs d'insert à l'aide de métriques d'analyse primaire

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0701000A2 (fr) * 1994-07-06 1996-03-13 New England Biolabs, Inc. DNA polymérase thermostable recombinante d'archaebactéries
WO2001023411A2 (fr) * 1999-09-30 2001-04-05 New England Biolabs, Inc. Incorporation de nucleotides modifies par des adn polymerases d'archaeon et methodes associees
WO2001032887A1 (fr) * 1999-10-29 2001-05-10 Stratagene Compositions et procedes utilisant des adn polymerases
WO2003054139A2 (fr) * 2001-06-29 2003-07-03 Stratagene Compositions et procedes utilisant des polymerases d'adn
WO2004039947A2 (fr) * 2002-08-19 2004-05-13 Stratagene Adn polymerases mutantes a activite transcriptase inverse

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997039150A1 (fr) 1996-04-15 1997-10-23 University Of Southern California Synthese d'adn marque par fluorophores
US6329178B1 (en) * 2000-01-14 2001-12-11 University Of Washington DNA polymerase mutant having one or more mutations in the active site
AU2007201277B2 (en) 2000-05-24 2011-10-13 Third Wave Technologies, Inc. Detection of RNA
EP1373889A2 (fr) 2000-07-31 2004-01-02 Maxygen, Inc. Biocapteurs, reactifs et applications diagnostiques d'evolution dirigee
CA2450139A1 (fr) 2001-06-11 2002-12-19 Illumina, Inc Techniques de detection multiplexees
ES2550513T3 (es) * 2002-08-23 2015-11-10 Illumina Cambridge Limited Nucleótidos modificados para secuenciación de polinucleótidos
GB0321306D0 (en) 2003-09-11 2003-10-15 Solexa Ltd Modified polymerases for improved incorporation of nucleotide analogues
US20070048748A1 (en) 2004-09-24 2007-03-01 Li-Cor, Inc. Mutant polymerases for sequencing and genotyping
US8623628B2 (en) 2005-05-10 2014-01-07 Illumina, Inc. Polymerases

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0701000A2 (fr) * 1994-07-06 1996-03-13 New England Biolabs, Inc. DNA polymérase thermostable recombinante d'archaebactéries
WO2001023411A2 (fr) * 1999-09-30 2001-04-05 New England Biolabs, Inc. Incorporation de nucleotides modifies par des adn polymerases d'archaeon et methodes associees
WO2001032887A1 (fr) * 1999-10-29 2001-05-10 Stratagene Compositions et procedes utilisant des adn polymerases
WO2003054139A2 (fr) * 2001-06-29 2003-07-03 Stratagene Compositions et procedes utilisant des polymerases d'adn
WO2004039947A2 (fr) * 2002-08-19 2004-05-13 Stratagene Adn polymerases mutantes a activite transcriptase inverse

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
GARDNER ANDREW F ET AL: "Acyclic and dideoxy terminator preferences denote divergent sugar recognition by archaeon and Taq DNA polymerases", NUCLEIC ACIDS RESEARCH, vol. 30, no. 2, 15 January 2002 (2002-01-15), pages 605 - 613, XP002309458, ISSN: 0305-1048 *
GARDNER ANDREW F ET AL: "Determinants of nucleotide sugar recognition in an archaeon DNA polymerase", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 27, no. 12, 15 June 1999 (1999-06-15), pages 2545 - 2553, XP002163101, ISSN: 0305-1048 *
LUTZ M J ET AL: "Recognition of a Non-Standard Base Pair by thermostable DNA Polymerases", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, OXFORD, GB, vol. 8, no. 10, 19 May 1998 (1998-05-19), pages 1149 - 1152, XP004137037, ISSN: 0960-894X *
QUN DONG ET AL: "Mutational studies of human DNA polymerase alpha: Identification of residues critical for deoxynucleotide binding and misinsertion fidelity of DNA synthesis", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 268, no. 32, 1993, pages 24163 - 24174, XP002309457, ISSN: 0021-9258 *
RODRIGUEZ A C ET AL: "Crystal structure of a pol alpha family DNA polymerase from the hyperthermophilic archaeon Thermococcus sp. 9<o>N-7", JOURNAL OF MOLECULAR BIOLOGY, LONDON, GB, vol. 299, no. 2, 2 June 2000 (2000-06-02), pages 447 - 462, XP004469030, ISSN: 0022-2836 *
SOUTHWORTH M W ET AL: "CLONING OF THERMOSTABLE DNA POLYMERASES FROM HYPERTHERMOPHILIC MARINE ARCHEA WITH EMPHASIS ON THERMOCOCCUS SP. 9 N-7 AND MUTATIONS AFFECTING 3'-5' EXONUCLEASE ACTIVITY", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE. WASHINGTON, US, vol. 93, no. 11, 28 May 1996 (1996-05-28), pages 5281 - 5285, XP000652319, ISSN: 0027-8424 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10519496B2 (en) 2001-12-04 2019-12-31 Illumina Cambridge Limited Labelled nucleotides
US9605310B2 (en) 2001-12-04 2017-03-28 Illumina Cambridge Limited Labelled nucleotides
US10480025B2 (en) 2001-12-04 2019-11-19 Illumina Cambridge Limited Labelled nucleotides
EP3002289B1 (fr) 2002-08-23 2018-02-28 Illumina Cambridge Limited Nucleotides modifies pour le sequençage de polynucleotide
US11008359B2 (en) 2002-08-23 2021-05-18 Illumina Cambridge Limited Labelled nucleotides
US10487102B2 (en) 2002-08-23 2019-11-26 Illumina Cambridge Limited Labelled nucleotides
US10513731B2 (en) 2002-08-23 2019-12-24 Illumina Cambridge Limited Modified nucleotides
US11028116B2 (en) 2003-08-22 2021-06-08 Illumina Cambridge Limited Labelled nucleotides
US10995111B2 (en) 2003-08-22 2021-05-04 Illumina Cambridge Limited Labelled nucleotides
US11028115B2 (en) 2003-08-22 2021-06-08 Illumina Cambridge Limited Labelled nucleotides
US10982277B2 (en) 2013-03-15 2021-04-20 Illumina Cambridge Limited Modified nucleosides or nucleotides
US10407721B2 (en) 2013-03-15 2019-09-10 Illumina Cambridge Limited Modified nucleosides or nucleotides
CN107922929A (zh) * 2015-09-09 2018-04-17 凯杰有限公司 聚合酶
US12104182B2 (en) 2018-12-05 2024-10-01 Illumina, Inc. Polymerases, compositions, and methods of use
WO2023020728A1 (fr) * 2021-08-14 2023-02-23 Illumina, Inc. Polymérases, compositions et procédés d'utilisation
US12077789B2 (en) 2021-08-14 2024-09-03 Illumina, Inc. Polymerases, compositions, and methods of use

Also Published As

Publication number Publication date
ATE532856T1 (de) 2011-11-15
US20160362664A1 (en) 2016-12-15
US20220010290A1 (en) 2022-01-13
US8460910B2 (en) 2013-06-11
JP5706057B2 (ja) 2015-04-22
GB0321306D0 (en) 2003-10-15
US20130244302A1 (en) 2013-09-19
EP1664287A1 (fr) 2006-06-07
US10017750B2 (en) 2018-07-10
EP2325304B1 (fr) 2012-05-09
US11136564B2 (en) 2021-10-05
US8852910B2 (en) 2014-10-07
DK2325304T3 (da) 2012-07-30
JP2007504817A (ja) 2007-03-08
US20220025342A1 (en) 2022-01-27
US11473067B2 (en) 2022-10-18
EP1664287B1 (fr) 2011-11-09
HK1156353A1 (en) 2012-06-08
US20060240439A1 (en) 2006-10-26
US9447389B2 (en) 2016-09-20
ATE557084T1 (de) 2012-05-15
WO2005024010A1 (fr) 2005-03-17
US20180298358A1 (en) 2018-10-18
US20150024463A1 (en) 2015-01-22

Similar Documents

Publication Publication Date Title
US11473067B2 (en) Modified polymerases for improved incorporation of nucleotide analogues
US10059928B2 (en) Polymerases
US8883454B2 (en) DNA polymerase fusions and uses thereof
EP1581630B1 (fr) Melanges d&#39;adn-polymerase et leurs utilisations
US9040276B2 (en) DNA binding protein-polymerase chimeras

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AC Divisional application: reference to earlier application

Ref document number: 1664287

Country of ref document: EP

Kind code of ref document: P

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PL PT RO SE SI SK TR

17P Request for examination filed

Effective date: 20111108

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AC Divisional application: reference to earlier application

Ref document number: 1664287

Country of ref document: EP

Kind code of ref document: P

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PL PT RO SE SI SK TR

REG Reference to a national code

Ref country code: GB

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 557084

Country of ref document: AT

Kind code of ref document: T

Effective date: 20120515

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 1156353

Country of ref document: HK

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602004037842

Country of ref document: DE

Effective date: 20120705

REG Reference to a national code

Ref country code: NL

Ref legal event code: T3

REG Reference to a national code

Ref country code: DK

Ref legal event code: T3

REG Reference to a national code

Ref country code: SE

Ref legal event code: TRGR

REG Reference to a national code

Ref country code: HK

Ref legal event code: GR

Ref document number: 1156353

Country of ref document: HK

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120509

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120509

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120509

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 557084

Country of ref document: AT

Kind code of ref document: T

Effective date: 20120509

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120910

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120810

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120509

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120509

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120509

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120509

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120509

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120509

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120509

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120509

PLBI Opposition filed

Free format text: ORIGINAL CODE: 0009260

PLAX Notice of opposition and request to file observation + time limit sent

Free format text: ORIGINAL CODE: EPIDOSNOBS2

26 Opposition filed

Opponent name: FROUD, CLIVE

Effective date: 20130208

Opponent name: KILGER, CHRISTIAN

Effective date: 20130206

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120930

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120820

REG Reference to a national code

Ref country code: DE

Ref legal event code: R026

Ref document number: 602004037842

Country of ref document: DE

Effective date: 20130206

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

PLAF Information modified related to communication of a notice of opposition and request to file observations + time limit

Free format text: ORIGINAL CODE: EPIDOSCOBS2

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120809

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120910

PLBB Reply of patent proprietor to notice(s) of opposition received

Free format text: ORIGINAL CODE: EPIDOSNOBS3

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20120509

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20120910

PLAB Opposition data, opponent's data or that of the opponent's representative modified

Free format text: ORIGINAL CODE: 0009299OPPO

R26 Opposition filed (corrected)

Opponent name: FROUD, CLIVE

Effective date: 20130208

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20040910

RDAF Communication despatched that patent is revoked

Free format text: ORIGINAL CODE: EPIDOSNREV1

APBM Appeal reference recorded

Free format text: ORIGINAL CODE: EPIDOSNREFNO

APBP Date of receipt of notice of appeal recorded

Free format text: ORIGINAL CODE: EPIDOSNNOA2O

APAH Appeal reference modified

Free format text: ORIGINAL CODE: EPIDOSCREFNO

APBQ Date of receipt of statement of grounds of appeal recorded

Free format text: ORIGINAL CODE: EPIDOSNNOA3O

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 13

APAH Appeal reference modified

Free format text: ORIGINAL CODE: EPIDOSCREFNO

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 14

REG Reference to a national code

Ref country code: FR

Ref legal event code: PLFP

Year of fee payment: 15

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: DK

Payment date: 20190910

Year of fee payment: 16

Ref country code: NL

Payment date: 20190912

Year of fee payment: 16

Ref country code: DE

Payment date: 20190827

Year of fee payment: 16

Ref country code: SE

Payment date: 20190910

Year of fee payment: 16

Ref country code: FR

Payment date: 20190815

Year of fee payment: 16

REG Reference to a national code

Ref country code: DE

Ref legal event code: R064

Ref document number: 602004037842

Country of ref document: DE

Ref country code: DE

Ref legal event code: R103

Ref document number: 602004037842

Country of ref document: DE

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20190906

Year of fee payment: 16

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: CH

Payment date: 20190913

Year of fee payment: 16

APBU Appeal procedure closed

Free format text: ORIGINAL CODE: EPIDOSNNOA9O

RDAG Patent revoked

Free format text: ORIGINAL CODE: 0009271

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: PATENT REVOKED

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

27W Patent revoked

Effective date: 20191126

GBPR Gb: patent revoked under art. 102 of the ep convention designating the uk as contracting state

Effective date: 20191126

REG Reference to a national code

Ref country code: FI

Ref legal event code: MGE

REG Reference to a national code

Ref country code: SE

Ref legal event code: ECNC